Novel reagents for transfection of eukaryotic cells

ABSTRACT

Compositions and methods for improved delivery of macromolecules into eukaryotic cells are provided. Fusogenic peptides from fusion proteins of non-enveloped viruses enhance the efficiency of transfection of eukaryotic cells mediated by transfection agents such as cationic lipids, polycationic polymers such as PEI and dendrimers. These fusogenic peptides are used as part of a transfection complex that efficiently delivers a macromolecule, for example, a nucleic acid, into a eukaryotic cell. Novel cationic lipids and compositions of cationic lipids also are provided that may be used for the introduction of macromolecules such as nucleic acids, proteins and peptides into a variety of cells and tissues. The lipids can be used alone, in combination with other lipids and/or in combination with fusogenic peptides to prepare transfection complexes.

This application claims priority to provisional application Ser. Nos.60/746,858 filed May 9, 2006; 60/746,854 filed May 9, 2006; 60/746,604filed May 5, 2006; 60/746,594 filed May 5, 2006; 60/746,424 filed May 4,2006; 60/771,865 filed Feb. 10, 2006; 60/771,864 filed Feb. 10, 2006;and 60/681,462 filed May 17, 2005. the contents of each of which arehereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Lipid aggregates such as liposomes can facilitate introduction ofmacromolecules, such as DNA, RNA, and proteins, into living cells.Aggregates comprising cationic lipid components can be used to effectdelivery of large anionic molecules, such as nucleic acids, into certaintypes of cells. See Feigner et al., Nature 337:387-388 (1989); Proc.Natl. Acad. Sci. USA 84:7413 (1987).

The use of cationic lipids has become increasingly popular since itsintroduction over 15 years ago. Several cationic lipids have beendescribed in the literature and some of these are commerciallyavailable. DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride) was the first cationic lipid to be synthesized for the purposeof nucleic acid transfection. See Feigner et al. (Proc. Nat'l Acad. Sci.84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated aloneor can be combined with DOPE (dioleoylphosphatidylethanolamine) into aliposome, and such liposomes can be used to deliver plasmids into somecells. Other classes of lipids subsequently have been synthesized byvarious groups. For example, DOGS(5-carboxyspermylglycinedioctadecylamide) was the first polycationiclipid to be prepared (Behr et al. Proc. Nat'l Acad. Sci. 86,6982 (1989);U.S. Pat. No. 5,171,678) and other polycationic lipids have since beenprepared. The lipid DOSPA(2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium)has been described as an effective delivery agent (U.S. Pat. No.5,334,761).

In other examples, cholesterol-based cationic lipids, such as DC-Chol(N,N-dimethyl-N-ethylcarboxamidocholesterol) have been prepared and usedfor transfection (Gao et al. Biochem. Biophys. Res. Comm. 179, 280(1991)). In another example 1,4-bis(3-N-oleylamino-propyl)piperazine wasprepared and combined with histone H1 to generate a delivery reagentthat was reported to be less toxic than other reagents (Wolf et al.BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335). Several reagentsare commercially available. Some exampics include Lipofectin®(DOTMA:DOPE) (Invitrogen, Carlsbad, Calif.), LipofectAmine™ (DOSPA:DOPE)(Invitrogen), LipofectAmine2000™ (Invitrogen) Fugene®, Transfectam®(DOGS), Effectene®, and DC-Chol. None of these reagents can be useduniversally for all cells. This is perhaps not surprising in light ofthe variation in composition of the membranes of different types ofcells as well as the barriers that can restrict entry of extracellularmaterial into cells. Moreover, the mechanism by which cationic lipidsdeliver nucleic acids into cells is not clearly understood. The reagentsare less efficient than viral delivery methods and are toxic to cells,although the degree of toxicity varies from reagent to reagent.

However, transfection agents, including cationic lipids, are notuniversally effective in all cell types. Effectiveness of transfectionof different cells depends on the particular transfection agentcomposition. In general, polycationic lipids are more efficient thanmonocationic lipids in transfecting eukaryotic cells. In many cases,cationic lipids alone are not effective or are only partially effectivefor transfection.

Many biological materials are taken up by cells via receptor-mediatedendocytosis, in which a ligand binds to a cell-surface receptor, leadingto clustering of ligand-bound receptors, and formation of coated pitsfollowed by internalization of the ligands into endosomes. Bothenveloped viruses, like influenza virus and alphaviruses, andnon-enveloped viruses, like Adenovirus, infect cells via endocytoticmechanisms. See: Pastan, I. et al. (1986) in “Virus Attachment and Entryinto Cells”, (Crowell, R. L. and Lonberg-Holm, K., eds.) Am. Soc.Microbiology, Washington, p. 141-146; Kielian et al., (1986) “Entry ofAlphaviruses” in The Togaviridae and Flaviviridae, (Schlesinger, S, andSchlesinger, M. J., eds.) Plenum Press, New York p. 91-119; FitzGeraldet al. (1983) Cell 32:607-617. Enhancement of dendrimer-mediatedtransfection of some cells by chloroquine (a lysosomotropic agent)suggests that endocytosis is involved in at least some transfections.

Introduction of foreign DNA sequences into eukaryotic cells mediated byviral infection is generally orders of magnitude more efficient thantransfection with anionic lipids, cationic lipid, PEI, peptides, ordendrimer transfection agents. Viral infection of all the cells in aculture requires fewer than 10 virus particles per cell. Although thedetailed mechanism of fusion is not fully understood and varies amongviruses, viral fusion typically involves specific fusogenic agents, suchas viral proteins, viral spike glycoproteins and peptides of viral spikeglycoproteins. Cell binding and internalization also can be enhanced,accelerated or made selective with peptides that bind cell receptors.For example, the penton-base protein of the Adenovirus coat contains thepeptide motif RGD (Arg-Gly-Asp) which mediates virus binding tointegrins and viral internalization via receptor-mediated endocytosis(Wickham et al. (1995) Gene Therapy 2:750-756).

The efficiency of cationic lipid transfections has been shown to beenhanced by the addition of whole virus particles to the transfectionmixture. Certain viral components may also enhance the efficiency ofcationic lipid-mediated transfection. For example, Kamata et al. ((1994)Nucl. Acids Res. 22:536) suggested that “Lipofectin™”-mediatedtransfections may be enhanced 3-4-fold by adding influenza virushemagglutinin peptides to the transfection mixture. Antibodies have beenshown to enhance cationic lipid transfections (Trubestsky, et al, (1992)BBA 1131, 311-313) and transferrin-poly lysine or asialoglycoproteinpolylysine have been shown to enhance cationic lipid transfection (Macket al, (1994) Am J Med. Sci. 138-143.

Nevertheless, these methods do not work for all cell types, requirerelatively complex protocols and are inconvenient. It is apparent,therefore, that new and improved methods for introducing macromolecules,and particularly nucleic acids, into cell, are greatly to be desired. Inparticular, improved methods for introducing nucleic acids into a widervariety of cells, and particularly into primary cells, are greatly to bedesired.

SUMMARY OF THE INVENTION

Disclosed herein are compositions and methods that provide improvedefficiency for introducing macromolecules, such as nucleic acids, intocells. Accordingly, provided herein is a complex containing a nucleicacid molecule, a transfection agent and a fusion agent, where the fusionagent contains a fusion-promoting amino acid sequence derived from afusion protein of a non-enveloped virus. The non-enveloped virus may bea Reovirus, for example, Avian Reovirus, Nelson Bay Reovirus, or PulauReovirus. In certain aspects, the complexes contain a macromolecule tobe introduced into the cell, such as a peptide, a protein, or a nucleicacid.

The fusion agent may contain a nucleic acid binding moiety functionallylinked to the fusion promoting amino acid sequence. Suitable nucleicacid binding moieties include a polycationic peptide sequence, apolyamine, a peptide nucleic acid, spermine, spermidine,carboxyspermidine and the like. The nucleic acid binding moiety may becovalently linked to the fusion promoting amino acid sequence. Thetransfection agent may be a cationic lipid, such as those describedbelow, a polyamine, a polycationic peptide sequence, or a cationicdendrimer or the like.

The fusion promoting amino acid sequence also may be functionally linkedto a lipid, such as a cationic or neutral lipid, and the linked moietymay be used for delivery of macromolecules into cells. For example, apeptide containing the fusion promoting amino acid sequence may becovalently linked to a lipid, such as a cationic lipid, using methodsthat are well known in the art.

The complex may also contain a transfection enhancing agent, such as anuclear localization protein or peptide, a fusogenic peptide or protein,receptor-ligand peptide or protein, a transport peptide or protein, or asecond viral peptide or protein that is distinct from the fusionpromoting amino acid sequence. The second viral peptide may be derivedfrom a virus such as an influenza virus, a vesicular stomatitis virus,an adenovirus, an alphavirus, a Semliki Forest Virus, a hepatitis virus,a herpes virus, an HIV virus, or a simian virus. The transfectionenhancing agent may also be, for example, insulin, a transferrin, aepidermal growth factor, a fibroblast growth factor, a cell targetingantibody, a lactoferrin, a fibronectin, an adenovirus penton base, Knob,a hexon protein, a vesicular stomatitis virus glycoprotein, a SemlikiForest Virus core protein, a influenza hemagglutinin, a hepatitis B coreprotein, an HIV Tat protein, a herpes simplex virus VP22 protein, ahistone protein, an arginine rich cell permeability protein, a highmobility group protein, and invasin protein, and internalin protein, anendotoxin, a diptheria toxin, a shigella toxin, a melittin, a magainin,a gramicidin, a cecrophin, a defensin, a protegrin, a tachyplesin, athionin, a indolicidin, a bactenecin, a drosomycin, an apidaecin, acathelicidin, a bacteriacidal-permability-increasing protein, a nisin, abuforin, or fragments thereof. The transfection enhancing agent may bechloroquine, a lysosomotrophic compound or combinations thereof. Thetransfection agent may contain multimers of the same or differentpeptides or proteins.

In particular embodiments, the transfection agent contains at least onecationic lipid, and may optionally also contain one or more neutrallipids. The cationic lipid may contain at least one monovalent cationiclipid or polycationic lipid, for example, DOSPA, DOSPER, DOGS, TMTPS,TMTOS, TMTLS, TMTMS, TMDOS.N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-hydroxypropane-1,3-diamine,N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-hydroxypropane-1,3-diamine,N-1-dimethyl-N-1-(2,3-diapalmityloxypropyl)-2-hydroxypropane-1,3-diamine,N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine,N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine,N-1-dimethyl-N-1-(2,3-diapalmityloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine,L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine,3,5-(N,N-di-lysyl)-diaminobenzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine),L-Lysine-bis(O,O′-oleoyl-β-hydroxyethyl)amide dihydrochloride,L-Lysine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride,1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl)piperazine,L-Lysine-bis-(O,O′-myristoyl-β-hydroxyethyl)amide dihydrochloride,L-Ornithine-bis-(O,O′-myristoyl-β-hydroxyethyl)amide dihydrochloride,L-Ornithine-bis-(O,O′-oleoyl-β-hydroxyethyl)amide dihydrochloride,1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxypropyl]piperazine,L-Ornithine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride,1,4,-bis[(3-amino-2-hydroxypropyl)-oleylamino]-butane-2,3-diol,1,4,-bis[(3-amino-2-hydroxypropyl)-palmitylamino]-butane-2,3-diol,1,4,-bis[(3-amino-2-hydroxypropyl)-myristylamino]-butane-2,3-diol,1,4-bis[(3-oleylamino)propyl]piperazine,L-Arginine-bis-(O,O′-oleoyl-β-hydroxyethyl)amide dihydrochloride,bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxypropyl]piperazine,L-Arginine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride,L-Serine-bis-(O,O′-oleoyl-β-hydroxyethyl)amide dihydrochloride,1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxypropyl]piperazine,Glycine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride,Sarcosine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride,L-Histidine-bis-(O,O′-palmitoyl-β-hydroxyethyl)amide dihydrochloride,cholesteryl-3β-carboxyl-amidoethylenetrimethylammonium iodide,1,4-bis[(3-myristylamino)propyl]piperazine,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylateiodide, cholesteryl-3β-carboxyamidoethyleneamine,cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinateiodide,2-[(2-trimethylammonio)-ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinateiodide, 3β[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol, and3β-[N-(polyethyleneimine)-carbamoyl]cholesterol,1,4-bis[(3-palmitylamino)propyl]piperazine,L-Ornithylglycyl-N-(1-heptadecyloctadecyl)glycinamide,N²,N⁵-Bis(3-aminopropyl)-L-ornithylglycyl-N-(1-heptadecyloctadecyl)glycinamide,1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazineN²-[N²,N⁵-Bis(3-aminopropyl)-L-ornithyl]-N,N-dioctadecyl-L-glutamine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dioctadecyl-L-α-glutamine,1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dioctadecyl-L-α-asparagine,N—[N²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N—N-dioctadecyl-L-glutaminyl]-L-glutamicacid, N²-[N²,N⁵-Bis(3-aminopropyl)-L-ornithyl]-N,N-diolyl-L-glutamine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dioleyl-L-α-glutamine,4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dioleyl-L-α-asparagine,N—[N²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N—N-dioleyl-L-glutaminyl-L-glutamicacid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine,N²-[N²,N⁵-Bis(3-aminopropyl)-L-ornithyl]-N,N-dipalmityl-L-glutamine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dipalmityl-L-α-glutamine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dipalmityl-L-α-asparagine,N—[N²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N—N-dipalmityl-L-glutaminyl]-L-glutamicacid,N²-[N²,N⁵-Bis(3-aminopropyl)-L-ornithyl]-N,N-dimyristyl-L-glutamine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dimyristyl-L-α-glutamine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dimyristyl-L-α-asparagine,1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)-2-hydroxypropyl]piperazine,N—[N²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N—N-dimyristyl-L-glutaminyl]-L-glutamicacid, 1,4-bis[(3-(3-aminopropyl)-myristylamino)propyl]piperazine,N²-[N²,N⁵-Bis(3-aminopropyl)-L-ornithyl]-N,N-dilaureyl-L-glutamine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dilaureyl-L-α-glutamine,N²-[N²,N⁵-Bis(aminopropyl)-L-ornithyl]-N—N-dilaureyl-L-α-asparagine,N—[N²-[N²,N⁵-Bis[(1,1-dimethylethoxy)carbonyl]-N²,N⁵-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N—N-dilaureyl-L-glutaminyl]-L-glutamicacid,3-[N′,N″-bis(2-tertbutyloxycarbonylaminoethyl)guanidino]-N,N-dioctadec-9-enylpropionamide,3-[N′,N″-bis(2-tertbutyloxycarbonylaminoethyl)guanidino]-N,N-dipalmitylpropionamide,3-[N′,N″-bis(2-tertbutyloxycarbonylaminoethyl)guanidino]-N,N-dimyristylpropionamide,1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine,1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl]piperazine,N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-diolylaminopropane,N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dipalmitylaminopropane,N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristylaminopropane,1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)propyl]piperazine,[(3-aminopropyl)-bis-(2-tetradecyloxyethyl)]methyl ammonium bromide,[(3-aminopropyl)-bis-(2-oleyloxyethyl)]methyl ammonium bromide,[(3-aminopropyl)-bis-(2-palmityloxyethyl)]methyl ammonium bromide,Oleoyl-2-hydroxy-3-N,N-dimethyamino propane,2-didecanoyl-1-N,N-dimethylaminopropane,palmitoyl-2-hydroxy-3-N,N-dimethyamino propane,1,2-dipalmitoyl-1-N,N-dimethylaminopropane,myristoyl-2-hydroxy-3-N,N-dimethyamino propane,1,2-dimyristoyl-1-N,N-dimethylaminopropane,(3-Amino-propyl)-)4-(3-amino-propylamino)-4-tetradecylcarbamoyl-butylcarbamicacid cholestryl ester,(3-Amino-propyl)-)4-(3-amino-propylamino-4-carbamoylbutylcarbamic acidcholestryl ester,(3-Amino-propyl)-4-(3-amino-propylamino)-4-(2-dimethylamino-ethylcarbamoy1)-butylcarbamic acid cholestryl ester, Spermine-5-carboxyglycine(N′-stearyl-N′-oleyl)amide tetratrifluoroacetic acid salt,Spermine-5-carboxyglycine (N′-stearyl-N′-elaidyl)amidetetratrifluoroacetic acid salt, Agmatinyl carboxycholesterol acetic acidsalt, Spermine-5-carboxy-β-alanine cholesteryl estertetratrifluoroacetic acid salt, 2,6-Diaminohexanoeyl β-alaninecholesteryl ester bistrifluoroacetic acid salt, 2,4-Diaminobutyroylβ-alanine cholesteryl ester bistrifluoroacetic acid salt,N,N-Bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesteryl estertristrifluoroacetic acid salt.,[N,N-Bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxy cholesteryl ester,Stearyl carnitine ester, Palmityl carnitine ester, Myristyl carnitineester, Stearyl stearoyl carnitine ester chloride salt, L-StearylStearoyl Carnitine Ester, Stearyl oleoyl carnitine ester chloride,Palmityl palmitoyl carnitine ester chloride, Myristyl myristoylcarnitine ester chloride, L-Myristyl myristoyl carnitine ester chloride,1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine,N-(3-aminopropyl)-N,N′-bis-(dodecyloxyethyl)-piperazinium bromide,N-(3-aminopropyl)-N,N′-bis-(oleyloxyethyl)-piperazinium bromide,N-(3-aminopropyl)-N,N′-bis-(palmityloxyethyl)-piperazinium bromide,N-(3-aminopropyl)-N,N′-bis-(myristyloxyethyl)-piperazinium bromide,N-(3-aminopropyl)-N-methyl-N,N′-(bis-2-dodecyloxyethyl)-piperaziniumbromide,N-(3-aminopropyl)-N′-methyl-N,N′-(bis-2-oleyloxyethyl)-piperaziniumbromide,N-(3-aminopropyl)-N′-methyl-N,N′-(bis-2-palmityloxyethyl)-piperaziniumbromide,N-(3-aminopropyl)-N′-methyl-N,N′-(bis-2-myristyloxyethyl)-piperaziniumbromide. The neutral lipids may be, for example DOPE, DPhPE, orcholesterol.

In other embodiments the transfection agent may contain involves atleast one polyamine transfection agent. Suitable polyamines includedense star dendrimers, PAMAM dendrimers, NH₃ core dendrimers,ethylenediamine core dendrimers, dendrimers of generation 5 or higher,dendrimers with substituted groups, dendrimers having one or more aminoacids, grafted dendrimers, activated dendrimers, polyethylenimine, andpolyethylenimine conjugates.

In specific embodiments, the fusion promoting amino acid sequence may becovalently linked to the transfection agents, the cationic lipid theneutral lipid, and/or the polyamine.

In other embodiments, the fusion promoting amino acid sequence may beconjugated to a nucleic acid binding group. The nucleic acid bindinggroup may be linked to a polyamine or peptide nucleic acid. Thepolyamine may contain at least one spermine moiety.

A complex as described above may contain two transfection agentsselected from the group consisting of fusogenic agents, nuclearlocalization sequences, transport peptides, receptor-ligand and a celladhesion peptide.

The invention further provides pharmaceutical compositions, containing acomplex as described above, and a pharmaceutical carrier.

The invention further provides methods of transfecting a cell, bycontacting a cell with a complex as described above. The cell may beprimary cell culture, a passaged cell culture or a cell line. Suitablecells include human cell lines and animal cell lines. The cell may be afibroblast.

In one method, a nucleic acid is contacted with a fusion agent and theresulting mixture is added to a mixture of a cationic lipid and aneutral lipid, where the fusion agent contains a fusion-promoting aminoacid sequence derived from a fusion protein of a non-enveloped virus.

In another method, a fusion agent is contacted with a transfection agentfollowed by addition of a nucleic acid or protein capable of aggregatingthe peptide- or protein-nucleic acid complex, where the fusion agentcontains a fusion-promoting amino acid sequence derived from a fusionprotein of a non-enveloped virus.

The invention further provides kits containing a transfection agent anda peptide or protein or a modified peptide or modified protein derivedfrom a fusion promoting amino acid sequence of Avian Reovirus, NelsonBay Reovirus, Pulau Reovirus or any Reovirus that may be capable ofenhancing transfection of the transfection agent. The kit may alsocontain a cationic lipid transfection agent. The cationic lipidtransfection agent may be selected from the group consisting ofLipofectAmine™ 2000, LipofectAmine™, Lipofectin®, DMRIE-C, CellFectin®(Invitrogen), Oligofectamine® (Invitrogen), LipofectAce® (Invitrogen),Fugene® (Roche, Basel, Switzerland), Fugene® HD (Roche), Transfectam®(Tranfectam, Promega, Madison, Wis.), Tfx-10® (Promega), Tfx-20®(Promega), Tfx-50® (Promega), Transfectin™ (BioRad, Hercules, Calif.),SilentFect™ (Bio-Rad), Effectene® (Qiagen, Valencia, Calif.), DC-chol(Avanti Polar Lipids), GenePorter® (Gene Therapy Systems, San Diego,Calif.), DharmaFect 1® (Dharmacon, Lafayette, Colo.), DharmaFect 2®(Dharmacon), DharmaFect 3® (Dharmacon), DharmaFect 4® (Dharmacon),Escort™ III (Sigma, St. Louis, Mo.) and Escort™ IV (Sigma)) The kit mayalso contain a polycationic polymer transfection agent, and also maycontain a diagnostic nucleic acid.

The present invention provides novel cationic lipids, and compositionsthat include such cationic lipids, that are useful for the delivery ofmacromolecules, such as nucleic acids, into cells. These novel cationiclipids have the structure according to Formula (I):

where X₁ and X₂ independently may be selected from the group consistingof (CH₂)_(n), (CHOH)_(n), and CONH; X₅ and X₆ independently are(CH₂)₁₋₆; W₁ and W₂ independently may be selected from the groupconsisting of hydrogen, —OH, —O—(C₁-C₃₀)alkyl, —O—(C₁-C₃₀) alkenyl,—O—(C₁-C₃₀)alkynyl, —NH₂, —NH(CH₂)_(s)CH₃, —N((CH₂)_(s)CH₃), —SH, and—NH—NH₂; R₃ and (R₆)_(q) independently may be selected from the groupconsisting of N, NH, CH, N(CH₂)_(s)CH₃, (CH)_(n), (COH)_(n), CON— andq=0-1; R₄ and R₅ independently may be selected from the group consistingof (CH₂)_(n), (CH₂—CHOH—CH₂)_(n), (CHOH)_(n), HNCO, CONH, CO, —O—, —S—,—S—S—, polyamide and an ester linkage; L₁ and L₂ independently may beselected from the group consisting of —NH—, —O—, —NHCO—, —CONH—, —OCO—,—COO—, —CO—, —S—, —S—S—, —NHC(O)O—, —OC(O)NH—, —NHCONH—, —NHC(═NH)NH—,—S(O)— and —SO₂—; Y is a heterocyclic moiety containing at least oneamine or amide moiety, where the points of attachment of Y are carbonand/or heteroatoms. Examples of suitable heterocyclic moieties include,but are not limited to, piperazine, piperidine, pyridine, pyrrolidine,and imidazole moieties and derivatives thereof. In specific embodiments,the heterocyclic moiety is a piperazine ring, where the points ofattachment optionally are at one or both of the nitrogen atoms. Theheterocyclic moiety may optionally be substituted with up to 4substituents independently selected from the group consisting of OH, ═O,a carboxylic acid, an ether, a polyether, an alkylaryl, an aminoalcohol, an amide, an straight chain alkyl, branched alkyl, cycloalkyl,straight chain alkenyl, branched alkenyl, cycloalkenyl, straight chainalkynyl, branched alkynyl, primary alkylamine, secondary alkylamine,tertiary alkyl amine, quaternary alkylamine, alkenylamine, secondaryalkenylamine, tertiary alkenyl amine, quaternary alkenylamine,alkynylamine, secondary alkynylamine, tertiary alkynylamine, quaternaryalkynylamine, amino alcohol, alcohol, ether, polyether, aryl, benzyl,heterocycle, cycloalkyl, alkyl polyamine, alkenyl polyamine, alkynylpolyamine, spermidine, spermine, carboxy spermine, guanidinium,pyridinium, pyrollidinium, piperidinium, piperazinium, and amino acyl,where the alkyl, alkenyl, alkynyl and alkylamine groups are optionallysubstituted with at least one hydroxyl, or at least one amine, or atleast one hydroxyl and at least one amine; R₁ and R₂ independently maybe selected from the group consisting of hydrogen, primary alkylamine,secondary alkylamine, tertiary alkyl amine, quaternary alkylamine,alkenylamine, secondary alkenylamine, tertiary alkenyl amine, quaternaryalkenylamine, alkynylamine, secondary alkynylamine, tertiaryalkynylamine, quaternary alkynylamine amino alcohol, alkyl polyamine,alkenyl polyamine, alkynyl polyamine, spermidine, spermine, carboxyspermine, guanidinium, pyridinium, pyrollidinium, piperidinium,piperazinium, amino acyl, peptidyl, and protein; Z₁ and Z₂ independentlymay be selected from the group consisting of straight chain alkyl,branched alkyl, cycloalkyl, straight chain alkenyl, branched alkenyl,cycloalkenyl, straight chain alkynyl, and branched alkynyl, m, n, p, ands independently are 0-6, with the proviso that when m, n, and p all are0 then Y is eliminated and R₃ is bonded directly to X₂. In oneembodiment, L₁ and L₂ independently may be selected from the groupconsisting of —NH—, —O—, —NHCO—, —CONH—, —NHC(O)O—, —OC(O)NH—, —NHCONH—,—NHC(═NH)NH—, —S(O)— and —SO₂—, and in another embodiment, L₁ and L₂independently may be selected from the group consisting of —NH—, —NHCO—,—CONH—, —NHC(O)O—, and —OC(O)NH—.

In accordance with one aspect of the invention, there is provided alipid having the structure according to Formula (II)

where X₁, X₂, W₁, W₂, R₃, (R₆), R₄, R₅, Y, R₁, R₂, Z₁, Z₂ m, n, p, and sare as defined above.

In a particular embodiment, Y may be

where X₃ and X₄ are independently selected from N and CH and where n₁and n₂ independently are 1-10. In any of the above embodiments, X₃ andX₄ are N and n₁ and n₂ independently are 1-10. For example, n₁ and n₂may be both 2. This cyclic structure may optionally be substituted withup to 4 substituents as defined above for Y.

In accordance with another aspect of the invention there is provided acomposition containing a lipid of Formula (I) and a co-lipid that isneutral, positively charged (such as a cationic lipid) or negativelycharged. The co-lipid may be, for example, DOPE or cholesterol. Thecationic lipid may include, but is not limited to, LipofectAmine™ 2000,LipofectAmine™, Lipofectin®, DMRIE-C, CellFectin®, Oligofectamine®,LipofectAce®, (Invitrogen) Fugene®, Fugene® HD (Roche), Transfectam®,Tfx-10®, Tfx-20,®Tfx-50® (Promega), Transfectin™, SilentFect™ (Bio-Rad),Effectene® (Qiagen), or DC-chol (Avanti Polar Lipids), GenePorter®(GTS), DharmaFect 1®, DharmaFect 2®, DharmaFect 3®, DharmaFect 4®(Dharmacon) Escort™ III or Escort™ IV (Sigma) The composition mayfurther contain a macromolecule, including, but not limited to, anucleic acid. Such nucleic acids can include, for example, DNA or RNA,either single stranded or double stranded (e.g. ssDNA, ssRNA, dsDNA, anddsRNA), and can include naturally occurring or non-naturally-occurringbases. The nucleic acid may be a plasmid, which may encode an RNAmolecule that is self complementary and that forms a region of doublestranded RNA. The nucleic acid may be an siRNA. Any of thesecompositions may further contain a eukaryotic cell, such as, by way ofexample only, a mammalian cell.

In accordance with yet another aspect of the invention there is provideda method of introducing a macromolecule into a cell, comprisingcontacting a eukaryotic cell with a composition as described above.

In accordance with another aspect of the invention there is provided acomposition comprising a lipid of Formula (I) as described herein, or acomposition comprising a lipid of Formula (I) and a co-lipid asdescribed above, and a peptide or protein. The peptide or protein may bea transfection enhancing peptide or protein that functions for nuclearor other sub-cellular localization, transport or trafficking. Thepeptide or protein may be transfection enhancing peptides or proteinsthat function as receptor ligands, that comprises a cell-adhesionsignal, a cell-targeting signal, a cell-internalization signal or anendocytosis signal, and combinations thereof. The peptide or protein maybe selected from the group consisting of peptides and proteins derivedfrom enveloped and non enveloped viruses, bacteria, insulin, atransferrin, an epidermal growth factor, a fibroblast growth factor, acell targeting antibody, a lactoferrin, a fibronectin, an adenoviruspenton base, Knob, a hexon protein, a vesicular stomatitis virusglycoprotein, a Semliki Forest Virus core protein, a influenzahemagglutinin, a hepatitis B core protein, an HIV Tat protein, a herpessimplex virus VP22 protein, a fusogenic peptide or protein, a reovirusfusion protein, a histone protein, an arginine-rich cell permeabilityprotein, a high mobility group protein, and invasin protein, andinternalin protein, an endotoxin, a diptheria toxin, a shigella toxin, amelittin, a magainin, a gramicidin, a cecrophin, a defensin, aprotegrin, a tachyplesin, a thionin, a indolicidin, a bactenecin, adrosomycin, an apidaecin, a cathelicidin, a adapatin protein, abacteriacidal-permability-increasing protein, a nisin, a buforin, andfragments thereof. These compositions may further contain amacromolecule, such as a nucleic acid, which may be a DNA molecule suchas a double stranded DNA molecule, optionally in the form of a plasmid.The plasmid may encode an RNA molecule that is self complementary andthat forms a region of double stranded RNA. The nucleic acid maycomprise an RNA molecule, such as a double stranded RNA molecule, forexample an siRNA. These compositions may be used to introduce amacromolecule, a peptide or a protein into a cell, by contacting aeukaryotic cell with a composition as described above. The peptide orprotein may be a transfection enhancing peptide or protein thatfunctions for nuclear or other sub-cellular localization, transport ortrafficking, is a receptor ligand, that comprises a cell-adhesionsignal, a cell-targeting signal, a cell-internalization signal or anendocytosis signal, and combinations thereof that is covalently modifiedwith spermine, spermidine or polylysine.

In accordance with another aspect of the invention there is provided amethod of introducing a desired molecule into a tissue, comprisingcontacting said tissue with a composition containing the desiredmolecule and a lipid or composition as described above. The desiredmolecule may be, for example, a nucleic acid, a peptide, or a protein.

In accordance with yet another aspect of the invention there is provideda kit for transfecting a cell, comprising a lipid of Formula (I).

In another aspect, the invention also provides complexes as describedabove where Y is

where X₃, X₄, n₁ and n₂ are as defined above. In these complexes, thecationic lipid may be a1,4-bis[(3-(3-aminopropyl)-alkylamino)propyl)piperazine lipid. In thecomplexes described above, the fusion promoting amino acid sequence maybe a peptide comprising 10-30 contiguous amino acids of a sequenceselected from the group consisting of:

MLRMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA,MPRMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA,MSGDCAGLVSVFGSVHCQSSKNKAGGDLQATSILTTYWPH,MSSDCAKIVSVFGSVHCQSSKNSAGGDLQATSVFTTYWPH,MGQRHSIVQPPAPPPNAFVEIVSSSTGIIIAVGIFAFIFS, andMGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI.

The peptide may contain at least 10 contiguous amino acids of an aminoacid sequence selected from the group consisting of:

RMPPGSCNGATAVFGNVH, GDCAGLVSVFGSVH, SDCAKIVSVFGSVH,QRHSIVQPPAPPPNAFVEIVS, and SGPSNFVNHAPGEAIVT,

covalently linked to between 8 and 30 lysine residues.

In the kits described above, a nucleic acid binding moiety and a peptideor protein or the modified peptide or modified protein derived from thefusion promoting amino acid sequence of a Reovirus, may be in the samecontainer. A nucleic acid binding moiety, a catioinic lipid transfectingagent, and a peptide or protein or a modified peptide or modifiedprotein derived from a fusion promoting amino acid sequence of aReovirus may be in the same container. The kits may also contain atransfection enhancing reagent. In one embodiment a kit contains anucleic acid binding moiety, a transfection enhancing reagent, acationic lipid transfecting agent, and a peptide or protein or amodified peptide or modified protein derived from a fusion promotingamino acid sequence of a Reovirus may be in the same container.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of transfection of CHO-K1 cells using thecomplexes of the invention using “before” and “after” protocols.

FIG. 2 shows the results of transfection of NIH-3T3 cells using thecomplexes of the invention using “before” and “after” protocols.

FIG. 3 shows the results of transfection of A549 cells using thecomplexes of the invention using “before” and “after” protocols.

FIG. 4 shows the results of transfection of COS cells using thecomplexes of the invention using “before” and “after” protocols.

FIG. 5 shows the results of transfection of BE2C cells using thecomplexes of the invention using “before” and “after” protocols.

FIG. 6 compares the results obtained from transfection of CHO cells withthe plasmid pCMV-Sport β-gal using LipofectAmine2000™, Transfectin™, andvarious formulations of a compound of the invention. Transfection wascarried out using a compound of Formula (I) formulated without aco-lipid (3-118-D) and with cholesterol in a molar ratio of 1:1(3-118-A), 2:1 (3-118-B) and 4:1 (3-118-C). Transfection withLipofectAmine™ 2000 (LA2K, Invitrogen Corp., Carlsbad, Calif.) andTransfectin™ (BioRad, Hercules, Calif.) also is shown for comparison.

FIG. 7 shows the results obtained from transfection of HEK 293 cellswith the plasmid pCMV-Sport β-gal using LipofectAmine™ 2000,Transfectin, and various formulations of a second compound of Formula(I). Transfection was carried out using a compound of the inventionformulated without a co-lipid (129E) and with cholesterol in M/M ratioof 1:1 (129H), 2:1 (129C) and 4:1 (129D); and a compound of thisinvention formulated with DOPE in M/M 2:1 (129A) and 4:1 (129B).Transfection with LipofectAmine™ 2000 (LA2K) and Transfectin™ is shownfor comparison

FIG. 8 shows the results obtained from transfection of NIH 3T3 cellswith the plasmid pCMV-Sport β-gal using LipofectAmine™ and DMRIE-C(Invitrogen Corp., Carlsbad, Calif.) and various formulations of asecond compound of Formula (I). Transfection was carried out using acompound of the invention formulated with cholesterol in M/M ratio of2:1 (129C); and compound of this invention formulated with DOPE in M/M2:1 (129A). Transfection with LipofectAmine™ and DMRIE-C is shown forcomparison.

FIG. 9 shows the results obtained from transfection of 293GT cells withthe plasmid pCMV-Sport β-gal using LipofectAmine™ and DMRIE-C(Invitrogen Corp., Carlsbad, Calif.) and various formulations of asecond compound of Formula (I) Transfection was carried out using acompound of the invention formulated with cholesterol in M/M ratio of2:1 (129C); and compound of this invention formulated with DOPE in M/M2:1 (129A). Transfection with LipofectAmine™ and DMRIE-C is shown forcomparison.

DETAILED DESCRIPTION

Compositions and methods for improved delivery of macromolecules intoeukaryotic cells are provided. The compositions and methods areeffective in a wide variety of cells, and provide a high efficiency oftransfection. Specifically, it has been found that fusogenic peptidesfrom fusion proteins of non-enveloped viruses can dramatically enhancethe efficiency of transfection of eukaryotic cells mediated bytransfection agents such as cationic lipids, polycationic polymers suchas PEI and dendrimers. These fusogenic peptides are used as part of atransfection complex that efficiently delivers a macromolecule, forexample, a nucleic acid, into a eukaryotic cell.

Novel cationic lipids and compositions of cationic lipids also areprovided that are effective for the introduction of macromolecules suchas nucleic acids, proteins and peptides into a variety of cells andtissues. The lipids can be used alone or in combination with otherlipids such as DOPE or cholesterol to form liposomes or lipid aggregatesthat are highly effective for delivery of macromolecules into cells invitro or in vivo. The lipids can also be used, for example, incombination with fusogenic peptides to prepare transfection complexes asdescribed herein. Methods for delivering macromolecules into targetcells and tissues using the lipids, alone or in combination, also areprovided.

The lipids have the following general structure (Formula I):

The skilled artisan will recognize that, although the molecules of theinvention are shown here for convenience in their neutral (unprotonated)forms, these molecules will exist in a partially or fully protonatedform in solutions of appropriate pH, and that the present inventionencompasses the molecules in all their protonated, unprotonated, ionizedand non-ionized forms without limitation, unless specifically indicatedotherwise.

In compounds of Formula I, X₁ and X₂ may independently be selected fromthe group consisting of (CH₂)_(n), (CHOH)_(n), and CONH. X₅ and X₆independently may be (CH₂)₁₋₆. W₁ and W₂ independently may be selectedfrom the group consisting of, hydrogen, —OH, —O—(C₁-C₃₀)alkyl,—O—(C₁-C₃₀)alkenyl, —O—(C₁-C₃₀)alkynyl, —NH₂, —NH(CH₂)_(s)CH₃,—N((CH₂)_(s)CH₃), —SH, and —NH—NH₂. R₃ and R₆ independently may beselected from the group consisting of N, NH, CH, N(CH₂)_(s)CH₃,(CH)_(n), (COH)_(n), CON— and q=0-1. R₄ and R₅ independently may beselected from the group consisting of (CH₂)_(n), (CH₂—CHOH—CH₂)_(n),(CHOH)_(n), HNCO, CONH, CO, —O—, —S—, —S—S—, polyamide and an esterlinkage. L₁ and L₂ independently may be selected from the groupconsisting of —NH—, —O—, —NHCO—, —CONH—, —OCO—, —COO—, —CO—, —S—, —S—S—,—NHC(O)O—, —OC(O)NH—, —NHCONH—, —NHC(═NH)NH—, —S(O)— and —SO₂—.

Y is a heterocyclic moiety containing at least one amine or amidemoiety. The points of attachment of Y may be carbon and/or heteroatoms.Examples of suitable heterocyclic moieties include, but are not limitedto, piperazine, piperidine, pyridine, pyrrolidine, and imidazolemoieties and derivatives thereof. In specific embodiments, theheterocyclic moiety is a piperazine ring, where the points of attachmentoptionally are at one or both of the nitrogen atoms. The heterocyclicmoiety may optionally be substituted with up to 4 substituentsindependently selected from the group consisting of OH, ═O, a carboxylicacid, an ether, a polyether, an alkylaryl, an amino alcohol, an amide,an straight chain alkyl, branched alkyl, cycloalkyl, straight chainalkenyl, branched alkenyl, cycloalkenyl, straight chain alkynyl,branched alkynyl, primary alkylamine, secondary alkylamine, tertiaryalkyl amine, quaternary alkylamine, alkenylamine, secondaryalkenylamine, tertiary alkenyl amine, quaternary alkenylamine,alkynylamine, secondary alkynylamine, tertiary alkynylamine, quaternaryalkynylamine, amino alcohol, alcohol, ether, polyether, aryl, benzyl,heterocycle, cycloalkyl, alkyl polyamine, alkenyl polyamine, alkynylpolyamine, spermidine, spermine, carboxy spermine, guanidinium,pyridinium, pyrollidinium, piperidinium, piperazinium, and amino acyl,where the alkyl, alkenyl, alkynyl and alkylamine groups are optionallysubstituted with at least one hydroxyl, or at least one amine, or atleast one hydroxyl and at least one amine,

R₁ and R₂ independently may be selected from the group consisting ofhydrogen, primary alkylamine, secondary alkylamine, tertiary alkylamine, quaternary alkylamine, alkenylamine, secondary alkenylamine,tertiary alkenyl amine, quaternary alkenylamine, alkynylamine, secondaryalkynylamine, tertiary alkynylamine, quaternary alkynylamine aminoalcohol, alkyl polyamine, alkenyl polyamine, alkynyl polyamine,spermidine, spermine, carboxy spermine, guanidinium, pyridinium,pyrollidinium, piperidinium, piperazinium, amino acyl, peptidyl, andprotein. In the context of the present invention it will be understoodthat, unless specifically indicated otherwise, an alkylamine can be anamine containing a short or a long alkyl chain. Similarly, analkenylamine will be understood to contain a short or long alkenylchain, and the same is true for alkynylamines.

Z₁ and Z₂ independently may be selected from the group consisting ofstraight chain alkyl, branched alkyl, cycloalkyl, straight chainalkenyl, branched alkenyl, cycloalkenyl, straight chain alkynyl,branched alkynyl where m, n, p, and s independently are 0-6, with theproviso that when m, n, and p all are 0 then Y is eliminated and R₃ isbonded directly to X₂.

Y may have the following cyclic structure

where X₃ and X₄ may independently be selected from N and CH and n₁ andn₂ independently are 1-10. Typically, Y is a 6-9 membered ring and, inexemplary specific embodiments, X₃ and X₄ are both N and n₁ and n₂ areboth 2, i.e. Y is an optionally substituted piperazine moiety. Thisstructure may optionally be substituted with 1-4 moieties as describedabove for Y.

In other specific embodiments Y can have the following cyclic structure:

where n₁, and n₂ independently are 1-10. Typically n₁+n₂ is 3-7. Such acyclic structure may optionally be substituted with 1-4 moietiesindependently selected as described above for Y.

Examples of the lipids may be defined by the following structure, whereL₁ and L₂ both are NH and X₅ and X₆ are CH₂:

In this structure R₁-R₆, W₁, W₂, X₁, X₂, Z₁, Z₂, Y m, p, and q are asdefined above.

A specific example of the lipids of the invention covered by the aboveformula is compound 5 given in example 1 below where R₁, R₂═H; X₁,X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=oleoyl; W₁, W₂═H; q, p,m=1; and Y=piperazine. Other specific examples include, but are notlimited to, the following:

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=palmityl;W₁, W₂═H; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=myristyl;W₁, W₂═H; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=lauryl; W₁,W₂═H; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=stearyl; W₁,W₂═H; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=oleoyl; W₁,W₂═OH; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=palmityl;W₁, W₂═OH; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=myristyl;W₁, W₂═OH; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=lauryl; W₁,W₂═OH; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=stearyl; W₁,W₂═OH; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=oleoyl; W₁,W₂═H; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=palmityl; W₁,W₂═H; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=myristyl; W₁,W₂═H; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=lauryl; W₁,W₂═H; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=stearyl; W₁,W₂═H; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=oleoyl; W₁,W₂═OH; q, p, m=1; and Y=piperazine:

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=palmityl; W₁,W₂═OH; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=myristyl; W₁,W₂═OH; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=lauryl; W₁,W₂═OH; q, p, m=1; and Y=piperazine;

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=stearyl; W₁,W₂═OH; q, p, m=1; and Y=piperazine.

The skilled artisan will recognize that the cationic lipids of thepresent invention are not limited to these specific examples.

In the context of the present invention, a short chain alkyl group istypically, unless otherwise defined, C₁-C₆ alkyl. A long chain alkylgroup is typically, unless otherwise defined, C₁₀-C₂₀ alkyl, or C₁₀-C₃₀alkyl. When not specifically defined, either definition may be used, asappropriate. The skilled artisan also will appreciate that otherderivative groups containing alkyl moieties, for example, alkoxymoieties and the like, also may contain short and/or long chain groupsas appropriate in the context, unless otherwise defined. An alkenylgroup contains at least one cis or trans carbon-carbon double bond andtypically is C₁₀-C₃₀ in chain length. Exemplary alkenyl groups containone or two cis double bonds where the double bonds are disubstituted. Analkynyl group contains at least one carbon-carbon triple bond andtypically is C₁₀-C₃₀ in chain length. The alkyl, alkenyl or alkynylgroups may be straight chain or branched. The skilled artisan also willappreciate that other derivative groups containing alkyl moieties, forexample, alkoxy moieties and the like, also may contain short and/orlong chain groups as appropriate in the context, unless otherwisedefined.

Lipids of the invention may be prepared by methods that are well knownin the art. See, for example, U.S. Pat. Nos. 5,334,761, 5,264,618,5,744,335, 5,527,928, WO00/27795 and Benerjee et al. (J. Med. Chem., 44,4176 (2001), each of which is hereby incorporated by reference in itsentirety.

Transfection Complexes

In one embodiment, a transfection complex provided herein contains themacromolecule that is to be delivered to the cell, a fusogenic peptide,and a transfection agent. The complex is formed and then added to thecells to be transfected. In other embodiments, where the macromoleculeis a nucleic acid, the fusogenic peptide is functionally linked to anucleic acid binding moiety. For example, the fusogenic peptide can belinked to a peptide sequence that binds nucleic acid, or to anotherpolycationic nucleic acid binding moiety as describe in more detailbelow. The functional linkage may be a covalent linkage or may benon-covalent. An example of a non-covalent linkage between the peptideand the nucleic acid binding moiety is where the peptide contains afirst member of a binding pair, and the nucleic acid binding moietycontains a second member of the binding pair, where association of thefirst and second members of the binding pair results in functionallinkage of the fusogenic peptide and the nucleic acid binding moiety.Suitable binding pairs include an antibody and an antigen,streptavidin/biotin, and the like. In certain illustrative aspects,transfection complexes are formed using complexes that include afusogenic peptide as disclosed herein, and a transfection reagent. Thesecomplexes that include a fusogenic peptide and a transfection reagentform another embodiment of the invention.

In still other embodiments, the complex may also contain a transfectionenhancing agent that facilitates entry of the complex into the targetcell or that facilitates subcellular or cellular targeting of thecomplex. Exemplary transfection enhancing agents include nuclearlocalization peptides, another fusogenic peptide or protein, a ligandfor a cell-surface receptor, and the like, as described in more detailbelow.

In other embodiments, the transfection complex of the invention containsthe macromolecule that is to be delivered to the cell and a fusogenicpeptide. Furthermore, in additional embodiments, provided herein is atransfection complex that includes a transfection agent and a fusogenicpeptide. Any of the transfection complexes provided herein can include atransfection enhancing agent, such as a nuclear localization sequence.Furthermore, the transfection complexes provided herein can include anucleic acid binding group.

Fusogenic peptides are provided that enhance transfection efficiency ofmacromolecules into cells. The peptides have amino acid sequences thatare derived from fusion proteins of non enveloped Reoviruses. Althoughthe skilled artisan will recognize that proteins, fragment thereof, ormodified peptides, proteins and fragments derived from fusion proteinsof a variety of non-enveloped viruses may be used in the presentinvention, it has been found that peptides from Avian Reovirus, NelsonBay Reovirus, and Pulau Reovirus are particularly useful. Thecompositions and methods of the invention, in illustrative embodiments,comprise peptides, proteins and fragments thereof, modified peptides,modified proteins and modified fragments thereof, peptide conjugates,protein conjugates and conjugates of fragments thereof, from suchnon-enveloped viruses.

These peptides are complexed with a transfection agent and amacromolecule, and the resulting complex is added to cells in culture,resulting in efficient intracellular delivery of the macromolecule. Thecomplexes and methods of the invention my be used to deliver a widevariety of macromolecules into cells but are particularly useful for thedelivery of nucleic acids. It will be understood that references todelivery of nucleic acid in the context of the present invention willalso convey to the skilled artisan that other macromolecules generallyalso can be used in place of the nucleic acid.

In other embodiments, other peptides, proteins, fragment thereof, ormodified peptides, proteins and fragments thereof that promote stillmore efficient transfection are used along with the complexes of theinvention. In one embodiment these peptides, proteins or fragmentsthereof are bound or added to the nucleic acid prior to adding thecomplex, while in other embodiments the peptides, proteins, or fragmentsthereof may be added or complexed with the complex prior to addition ofthe nucleic acid. Alternatively, the nucleic acid may be combined withthe complex prior to addition of the peptide, protein, etc.

Fusogenic Peptides

The present inventors have surprisingly found that amino acid sequencesderived from non-enveloped viruses are highly efficient at promotingtransfection of macromolecules into cells, including into cells such asprimary cells, that are otherwise refractory to common transfectionagents. Advantageously, the peptides have sequences that are derivedfrom the N-terminal regions of fusion-associated small transmembrane(FAST) proteins encoded by non-enveloped fusogenic reoviruses.

These reoviruses enter cells by membrane fusion followed by syncytiumformation. It is thought that syncytium formation is mediated by smallnon-structural transmembrane proteins, sometimes referred to as FASTproteins, that localize to the surface of the target cell and induceefficient cell-cell fusion. Shmulevitz et al., EMBO J. 19:902 (2000);Cheng et al., J. Virol., 79:1853 (2005). Surprisingly, the presentinventors have found that short peptides derived from these fusogenicproteins efficiently promote transfection of artificial complexescontaining nucleic acids into cells.

Certain peptides derived from these fusogenic proteins are believed tobe fusogenic peptides that provide illustrative examples of fusionpromoting amino acid sequences according to the present invention. Afusogenic peptide included in the compositions and methods providedherein, can be derived from any fusogenic protein of a non-envelopedvirus. Advantageously, the peptide comprises an amino acid sequencederived from the N-terminus of the fusogenic protein. Typically thepeptide is derived from approximately the first 50 amino acids of thenative fusogenic protein sequence, and advantageously contains 15-25amino acids derived from the N-terminus of the fusogenic protein,although longer or shorter sequences also can be used. Isolatedfusogenic peptides of Reoviruses provided herein, themselves form aseparate embodiment of the invention.

Reovirus FAST proteins typically contain a conserved N-terminal domainstructure comprising, in N-terminal to C-terminal order, a hydrophobicregion, a transmembrane region and a polybasic region. In some virusesthe hydrophobic region contains or is replaced by a polyproline motif.The N-terminus is optionally myristoylated. The peptides that are usefulfor the present invention typically are derived from the hydrophobicregion prior to the transmembrane domain, although part or all of thetransmembrane domain may be included in the sequence. Methods ofidentifying transmembrane domains are well known in the art. See, forexample, White, Annu. Rev. Physiol., 52:675-697 (1990). A typicaltransmembrane domain is a contiguous sequence of amino acids averaging29 residues, with average hydrophobicity of 0.7±0.09 and analanine+glycine content of 16%±8%.

The peptide can be myristoylated at the N-terminus although this is notrequired for efficient transfection. This is surprising because it hasbeen reported that the presence of a fatty acid moiety is essential formembrane fusion in avian reovirus and Nelson Bay reovirus.

Shmulevitz et al., J. Virol. 77:9769 (2003). The fusion promoting aminoacid sequence also may be functionally linked to a lipid, such as acationic or neutral lipid, and the linked moiety may be used fordelivery of macromolecules into cells. For example, a peptide containingthe fusion promoting amino acid sequence may be covalently linked to alipid, such as a cationic lipid, using methods that are well known inthe art.

It has also been described that reovirus FAST proteins contain aconserved region between the hydrophobic region and the transmembranedomain. See Shmulevitz et al., J. Virol. 77:9769 (2003). The peptidesthat are useful in the context of the present invention optionally mayinclude amino acids from this conserved region; however, in the examplesprovided below, the peptides lack these amino acids.

The Nelson Bay Reovirus FAST fusogenic protein has the followingsequence:

 1 MSSDCAKIVS VFGSVHCQSS KNSAGGDLQA TSVFTTYWPH FAIGGGIIVV 51ILLLGLFYCC YLKWKTSQVK HTYRRELIAL TRSHVHSTPS GISYV

The complete sequence of the Avian reovirus FAST fusogenic protein is:

MLRMPPGSCN GATAI/VFGNVH CQAAQNTAGG DLQATSSIIAYWPYLAAGGG FLLIVIIFAI LYCCKAKVKA DAARSVFHRE LVALSSGKHN AMAPPYNV

Prototypical peptides that are useful in the context of the presentinvention that are derived from this protein have the sequences providedbelow in the section entitled Protypical Avian reovirus fusogenicpeptides.

The skilled artisan will recognize that other peptides can be derivedfrom the fusion protein sequence that can be used in the presentinvention. In particular, peptides containing conservative amino acidsubstitutions may be used. Conservative amino acid substitutions arewell known in the art. Typical substitutions can be made where the aminoacids are similar in size and/or charge properties. For example, lysineand arginine, aspartate and glutamate and isoleucine and valine arepairs of similar amino acids. Methods of determining similarity betweenamino acid pairs have been described using a number of methods. Forexample, Dayhoff et al. (1978) in Atlas of Protein Sequence andStructure, Volume 5, Supplement 3, Chapter 22, pages 345-352, which isincorporated by reference herein, provides frequency tables for aminoacid substitutions which can be employed as a measure of amino acidsimilarity. In addition, since the peptides are most convenientlyproduced by chemical peptide synthesis, non-naturally occurring aminoacids can be substituted using known substitution patterns. For example,2-amino-5-hexanoic acid can be used in place of methionine.

The present invention also includes peptides having defined sequenceidentities with the N-terminal region of reovirus fusogenic proteins. Inparticular, these peptides in illustrative embodiments, contain no morethan about 25 contiguous amino acids of the N-terminal region sequenceof a reovirus fusogenic protein, prior to the reovirus protein conservedregion and the transmembrane domain. The peptides typically contain asequence that is derived at least in part from the N-terminalhydrophobic domain of the fusogenic peptide sequence. The diagram belowshows the approximate location of the hydrophobic (bold), conserved(underlined) and transmembrane (double underlined) domains for avianreovirus:

MLRMPPGSCNGATAIFGNVHCQAAQNTAGG DLQATSSIIAYWPYLAAGGGFLLIVIIFAILYCCKAKVKA DAARSVFHRE LVALSSGKHN AMAPPYNV

The skilled artisan will recognize that in certain embodiments, peptidescan be used that are at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,and 20 amino acids, and no more than 75, 70, 60, 50, 40, 30, or 25 aminoacids in length having a region of greater than or equal to 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 45, 50or all amino acids of the amino terminal 50 amino acids of a Reovirusfusogenic FAST protein, for example an avian Reovirus fusogenic FASTprotein or peptide derived therefrom, such as from an avian reoviruspeptide having the sequence RMPPGSCN GATAIFGNVH. In another embodimentof the present invention, a fusogenic peptide of a fusion agent, has asequence that is between 5 and 50 amino acids in length and includes atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids of a hydrophobicregion of a Reovirus FAST protein and can optionally include all or aportion of the conserved region between the hydrophobic region and thetransmembrane domain. Sequence identity can be calculated using, forexample, sequence analysis software such as the Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705. Forexample, amino acid sequences may be aligned to maximize identity using,if necessary, gaps to produce appropriate alignment. The percentageidentity is calculated by recording all of the positions in which theamino acids of both sequences are identical, relative to the totalnumber of positions.

The Nelson Bay Reovirus fusion protein, from which fusogenic peptides ofthe present invention can be derived, has the following sequence:

 1 MSSDCAKIVS VFGSVHCQSS KNSAGGDLQA TSVFTTYWPH FAIGGGIIVV 51ILLLGLFYCC YLKWKTSQVK HTYRRELIAL TRSHVHSTPS GISYV

The hydrophobic region of the protein is shown in bold. Peptides derivedfrom the first 1-50, advantageously the first 1-40, more advantageouslythe first 1-25 amino acids of this protein, can be used in the presentinvention. In an alternative embodiment, peptides that are 5 to 50 aminoacids in length that include at least 5, 6, 7, 8, 9, 10, 11, 12, 13, orall amino acids of the hydrophobic region of a Reovirus are used. Inanother embodiment Advantageously, the peptides contain most or all ofthe hydrophobic region of the protein as shown above. Similarly,N-terminal peptides derived from the baboon reovirus or the reptilianreovirus can be used. See Corcoran et al., J. Virol., 78:4342 (2004),and Dawe et al. J. Virol., 76:2131 (2002), the contents of each of whichare hereby incorporated by reference in their entireties. Peptidesderived from the N-terminal region of the fusogenic peptides from otherreoviruses, whether presently known or discovered in the future, can beused in the present invention. Specific further examples includepeptides derived from the fusogenic protein of the Muscovy duckreovirus.

In certain embodiments, the fusogenic peptide of the present inventionis 5 to 50 amino acids in length, preferably 10 to 40 amino acids inlength, and includes at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14contiguous amino acids that are at least 80, 85, 90, 95, 99, or 100%identical to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acidsof the hydrophobic region of a Reovirus FAST protein, in illustrativeexamples an Avian Reovirus FAST protein. The fusogenic peptide incertain illustrative examples includes a polycationic peptide sequence.

Prototypical Avian Reovirus Fusogenic Peptides

The specific examples of this section provide Prototypical Avianreovirus fusogenic peptides of the present invention.

Specific examples of peptides according to the invention include, butare not limited to, peptides that are 5-50 amino acids in length andthat contain 5-30 contiguous amino acids from one of the followingsequences:

MLRMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA,MLRMPPGSCNG TAIFGNVHCQAAQNTAGGDLQATSSIIA,MPRMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA,MSGDCAGLVSVFGSVHCQSSKNKAGGDLQATSILTTYWPH,MSSDCAKIVSVFGSVHCQSSKNSAGGDLQATSVFTTYWPH,MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI,MGQRHSIVQPPAPPPNAFVEIVSSSTGIIIAVGIFAFIFS,MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI.

Further specific examples of peptides according to the inventioninclude, but are not limited to, peptides that contain at least 5contiguous amino acids from one of the following sequences:

RMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA,RMPPGSCNGATAIFGNVHCQAAQNTAGGDLQATSSIIA,GDCAGLVSVFGSVHCQSSKNKAGGDLQATSILTTYWPH,SDCAKIVSVFGSVHCQSSKNSAGGDLQATSVFTTYWPH,SGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI,QRHSIVQPPAPPPNAFVEIVSSSTGIIIAVGIFAFIFS,SGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI.

Further specific examples of peptides according to the inventioninclude, but are not limited to, peptides that contain 5-all contiguousamino acids from one of the following sequences:

RMPPGSCNGATAVFGNVH, RMPPGSCNGATAIFGNVH, GDCAGLVSVFGSVHCQSS,SDCAKIVSVFGSVHCQSS, QRHSIVQPPAPPPNAFVE, SGPSNFVNHAPGEAIVTG.

These peptides can be linked at either terminus to a polycationicpeptide sequence or other nucleic acid binding moiety. The inventionalso includes variants on these peptides that have at least about 50,60, 70, 75, 80, 85, 90, 95 or 99% sequence identity to one of thepeptides.

These fusogenic peptides can be used directly in this form for preparingthe transfection complexes of the present invention. Advantageously,however, the fusogenic peptides are linked to a nucleic acid bindingmoiety (or other macromolecule binding moiety) to facilitate efficienttransfection. The nucleic acid binding moiety can be any of the manyknown moieties that are used for binding to nucleic acids.Advantageously the binding moiety is cationic or polycationic such thatit binds via electrostatic attraction to the polyanionic nucleic acid.Suitable nucleic acid binding moieties include polycationic peptides,such as peptides containing a high percentage of lysine and/or arginineresidues, polyamines such as spermine or spermidine or the like, andpeptide nucleic acids. Other nucleic acid binding moieties are wellknown in the art.

The fusogenic peptide is functionally linked to the nucleic acid bindingmoiety such that when a transfection complex is formed the nucleic acidthat is to be transfected is associated with the fusogenic peptidesufficiently that the transfection is enhanced over that observed in theabsence of the fusogenic peptide. This is advantageously achieved bycovalently linking the fusogenic peptide to the nucleic acid bindingdomain, although a non-covalent association also can be used. To achievenon-covalent association of the fusogenic peptide and the nucleic acidbinding domain, for example, the peptide and the binding domain canrespectively be covalently linked to a member of a specific bindingpair. For example, the fusogenic peptide can be coupled to biotin andthe binding domain could be coupled to streptavidin. Alternatively, thepeptide can be linked to a hapten and the binding domain can be coupledto an antibody or antibody fragment, such as an scFv.

Advantageously, however, the fusogenic peptide and the nucleic acidbinding domain are covalently linked Suitable linkers for achieving suchlinkages are well known in the art and are commercially available, forexample, from Pierce (Rockford, Ill.). The fusogenic peptide can bederivatized with a bifunctional linker during peptide synthesis and thislinker can then be used to form a covalent bond to the nucleic acidbinding domain. Alternatively, a nucleophilic amino acid derivative canbe introduced into the fusogenic peptide sequence during synthesis, andthis derivative can be used to couple the peptide to the nucleic acidbinding domain.

Most conveniently, the fusogenic peptide is covalently linked to apolycationic peptide sequence during peptide synthesis. Suitablepolycationic peptide sequences contain multiple lysine, ornithine and/orarginine residues, although other basic amino acids also can be usedincluding non-naturally occurring amino acids. Typically, a polycationicpeptide will contain 10-30 lysine, ornithine, or arginine residues, andwill be 5 to about 50 amino acids long, although the skilled artisanwill recognize that any peptide sequence that electrostatically binds tonucleic acids can be used. Advantageously, the polycationic peptidecontains 15-20 basic residues and is 15-30 amino acids long.

Specific examples of peptides according to the invention include, butare not limited to, peptides that are 20 to 50 amino acids long and thatcontain 5-30 contiguous amino acids from one of the following sequences,covalently linked to between 15 and 20 contiguous lysine residues:

MLRMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA,MPRMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA,MSGDCAGLVSVFGSVHCQSSKNKAGGDLQATSILTTYWPH,MSSDCAKIVSVFGSVHCQSSKNSAGGDLQATSVFTTYWPH,MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI,MGQRHSIVQPPAPPPNAFVEIVSSSTGIIIAVGIFAFIFS,MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI.

Further specific examples of peptides according to the inventioninclude, but are not limited to, peptides that contain 5-30 contiguousamino acids from one of the following sequences, covalently linked tobetween 15 and 20 contiguous lysine residues:

RMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA,RMPPGSCNGATAIFGNVHCQAAQNTAGGDLQATSSIIA,GDCAGLVSVFGSVHCQSSKNKAGGDLQATSILTTYWPH,SDCAKIVSVFGSVHCQSSKNSAGGDLQATSVFTTYWPH,SGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI,QRHSIVQPPAPPPNAFVEIVSSSTGIIIAVGIFAFIFS,SGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI.

Further specific examples of peptides according to the inventioninclude, but are not limited to, one of the following sequences:

RMPPGSCNGATAVFGNVHKKKKKKKKKKKKKKKK RMPPGSCNGATAIFGNVHKKKKKKKKKKKKKKKKGDCAGLVSVFGSVHCQSSKKKKKKKKKKKKKKKK, SDCAKIVSVFGSVHCQSSKKKKKKKKKKKKKKKK,QRHSIVQPPAPPPNAFVEKKKKKKKKKKKKKKKK, SGPSNFVNHAPGEAIVTGKKKKKKKKKKKKKKKK.

These peptides contain 16 lysine residues that confer the ability tobind to nucleic acids on the peptide. It will be recognized that thepresent invention also includes peptides where some or all of theselysine residues are replaced by other positively charged amino acidresidues.

Transfection Agents

An additional component of the complexes used in the present inventionis a transfection agent. Suitable transfection agents in the context ofthe present invention include cationic and polycationic polymers, and/orcationic and polycationic lipids. Cationic and polycationic polymerssuitable for use in the invention are known in the art and include, forexample, dense star dendrimers, PAMAM dendrimers, NH₃ core dendrimers,ethylenediamine core dendrimers, dendrimers of generation 5 or higher,dendrimers with substituted groups, dendrimers comprising one or moreamino acids, grafted dendrimers and activated dendrimers,polyethyleneimine, polyethyleneimine conjugates, and polyalkylenimine.The skilled artisan will recognize that the present invention is notlimited to use of these polycationic polymer transfection agents.

Advantageously, the transfection agent is a lipid, preferably a cationiclipid (or a mixture of a cationic lipid and neutral lipid). This lipidcan be used to form a peptide- or protein-nucleic acid-lipid aggregatewhich facilitates introduction of the anionic nucleic acid through cellmembranes, including the nuclear membrane. Transfection compositions ofthis invention comprising peptide- or protein-nucleic acid complexes andlipid can further include other non-peptide agents that are known tofurther enhance transfection.

Inclusion of a peptide- or protein-nucleic acid complex or a modifiedpeptide- or protein-nucleic acid complex in a cationic lipidtransfection composition can significantly enhance transfection (by2-fold or more) of the nucleic acid compared to transfection of thenucleic acid mediated by the cationic lipid alone. Enhancement ofpolycationic polymer transfection by peptides or proteins or modifiedpeptides or modified proteins or fragments thereof of the presentinvention is pronounced in a wide variety of cell lines, including humanprimary cell lines and in cell lines that are generally considered bythose in the art to be “hard-to-transfect.”

Monovalent or polyvalent cationic lipids are employed in cationic lipidtransfecting compositions. Illustrative monovalent cationic lipidsinclude DOTMA (N-[1-(2.3-dioleoyloxy)-propyl]-N,N,N-trimethyl ammoniumchloride), DOTAP (1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane),DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammoniumbromide), DDAB (dimethyl dioctadecyl ammonium bromide), DC-Chol(3-(dimethylaminoethane)-carbamoyl-cholestrerol). Preferred polyvalentcationic lipids are lipospermines, specifically, DOGS(Dioloctadecylaminoglycyl spermine), DOSPA(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-iumtrifluoro-acetate) and DOSPER (1,3-dioleoyloxy-2-(6-carboxyspermyl)-propyl-amid;N-1-dimethyl-N-1-(2,3-dialkyloxypropyl)-2-hydroxypropane-1,3-diamineincluding but not limited toN-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-hydroxypropane-1,3-diamine,N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-hydroxypropane-1,3-diamine,N-1-dimethyl-N-1-(2,3-diapalmityloxypropyl)-2-hydroxypropane-1,3-diamine;N-1-dimethyl-N-1-(2,3-dialkyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamineincluding but not limited toN-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine,N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine,N-1-dimethyl-N-1-(2,3-diapalmityloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine;and the di- and tetra-alkyl-tetra-methyl spermines, including but notlimited to TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS(tetramethyltetraoleyl spermine), TMTLS (tetramethlytetralaurylspermine), TMTMS (tetramethyltetramyristyl spermine) and TMDOS(tetramethyldioleyl spermine); and1,4,-bis[(3-amino-2-hydroxypropyl)-alkylamino]-butane-2,3-diol includingbut not limited to1,4,-bis[(3-amino-2-hydroxypropyl)-oleylamino]-butane-2,3-diol,1,4,-bis[(3-amino-2-hydroxypropyl)-palmitylamino]-butane-2,3-diol,1,4,-bis[(3-amino-2-hydroxypropyl)-myristylamino]-butane-2,3-diol; and1,4-bis(3-alkylaminopropyl)piperazine including but not limited to1,4-bis[(3-oleylamino)propyl]piperazine,1,4-bis[(3-myristylamino)propyl]piperazine,1,4-bis[(3-palmitylamino)propyl]piperazine; and a1,4-bis[(3-(3-aminopropyl)-alkylamino)propyl)piperazine including butnot limited to 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine,1,4-bis[(3-(3-aminopropyl)-myristylamino)propyl]piperazine,1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine; and1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)propyl]piperazineincluding but not limited to1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl]piperazine,1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)propyl]piperazine,1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine,1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxy-propyl]piperazineincluding but not limited to1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine,1,4-bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxy-propyl]piperazine,1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]piperazine

In certain illustrative examples the cationic lipid is a lipid ofFormula (I) such as a1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxy-propyl]piperazine, asdescribed in more detail below. Other cationic lipids that may be usedinclude the commercial agents LipofectAmine™ 2000, LipofectAmine™,Lipofectin®, DMRIE-C, CellFectin® (Invitrogen), Oligofectamine®(Invitrogen), LipofectAce® (Invitrogen), Fugene® (Roche, Basel,Switzerland), Fugene® HD (Roche), Transfectam® (Tranfectam, Promega,Madison, Wis.), Tfx-10® (Promega), Tfx-20® (Promega), Tfx-50® (Promega),Transfectin™ (BioRad, Hercules, Calif.), SilentFect™ (Bio-Rad),Effectene® (Qiagen, Valencia, Calif.), DC-chol (Avanti Polar Lipids),GenePorter® (Gene Therapy Systems, San Diego, Calif.), DharmaFect 1®(Dharmacon, Lafayette, Colo.), DharmaFect 2® (Dharmacon), DharmaFect 3®(Dharmacon), DharmaFect 4® (Dharmacon), Escort™ III (Sigma, St. Louis,Mo.) and Escort™ IV (Sigma).

Cationic lipids are optionally combined with non-cationic lipids,particularly neutral lipids, for example lipids such as DOPE(dioleoylphosphatidylethanolamine), DPhPE(diphytanoylphosphatidylethanolamine) or cholesterol. The ratio can varyfrom 1:1 (molar) to 4:1 (molar) of cationic to neutral lipids.Transfection properties of cationic lipid compositions composed of a 1:1to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine and DOPE and a1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine and cholesterolas well as a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine and DOPE anda 1:1 to 4:1 mixture of1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine andcholesterol are significantly enhanced by peptides and proteins of theinvention.

Transfection properties of cationic lipid compositions composed of a 1:1to 4:1 mixtures of1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl]piperazine andDOPE and a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl]piperazine andcholesterol as well as a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine andDOPE and a 1:1 to 4:1 mixture of1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine andcholesterol are significantly enhanced by peptides and proteins of theinvention.

A cationic lipid composition composed of a 3:1 (w/w) mixture of DOSPAand DOPE or a 1:1 (w/w) mixture of DOTMA and DOPE is generally useful intransfecting compositions of this invention, although it will beappreciated that many other compositions can be used. Preferredtransfection compositions are those which induce substantialtransfection of a higher eukaryotic cell line. Inclusion of a peptide-or protein-nucleic acid or modified peptide- or protein-nucleic acidcomplex in a polycationic polymer transfection composition cansignificantly enhance transfection (by 2-fold or more) of the nucleicacid compared to transfection of the nucleic acid mediated by thepolycationic polymer (e.g. dendrimer) alone or in combination withDEAE-dextran or chloroquine or both. Enhancement of transfection bypeptides, proteins, modified peptides or modified proteins is pronouncedin a wide variety of cell lines, including human primary cell lines andin cell lines that are generally considered by those in the art to be“hard-to-transfect.”

Preparation of the Lipids of Formula (I):

The lipids of Formula I can be synthesized as described generally belowand as described in more detail in the examples. Those skilled in theart will recognize that other members of these classes of lipids can besynthesized using variations of these methods or other methods that arewell known in the art.

An amine-containing cyclic moiety such as1,4-bis(3-amino-2-hydroxypropyl)piperazine may be prepared, for example,by alkylation of piperazine with N-(2,3-epoxypropyl)phthalimide,followed by removal of the phthalamide group using hydrazine hydrate.The resulting 1,4-bis(-3-amino-2-hydroxypropyl)piperazine may beacylated with an activated carboxyl compound, for example and alkyl acidchloride or alkenyl acid chloride such as oleoyl chloride. The resultingamide may be reduced, for example with lithium aluminum hydride, and theresulting secondary amine alkylated using a haloalkylphthalimide, suchas 3-bromopropylpthalimide. The phthalimide moiety may be removed using,for example, hydrazine hydrate and the resulting amine may be protonatedwith an acid such as HCl or trifluoroacetic acid to obtain the desiredcationic lipids. The skilled artisan will recognize that this generalreaction scheme can be used to prepare a wide variety of cationic lipidsof the present invention. For example,1,4-bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine or1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl]piperazine, wherealkyl can constitute a C₁₀-C₃₀ alkyl chain, can be synthesized.Substituted heterocyclic rings can be prepared using methods that arewell known in the art. For example, disubstituted piperazine moietiesmay be prepared from diketopiperazine compounds by reduction of thelactam groups with a suitable reducing agent, as described, for example,in J. Med. Chem. 39:1345 (1996).

Transfection Enhancing Agents

The complexes formed between the fusogenic peptide, the optional nucleicacid binding domain, the nucleic acid and the transfection agent may befurther enhanced by inclusion of moieties such as proteins or peptidesthat function for nuclear or other sub-cellular localization, functionfor transport or trafficking, are receptor ligands, comprisecell-adhesive signals, cell-targeting signals, cell-internalizationsignals or endocytosis signals as well as peptides or functionalportions thereof of viral fusogenic proteins of enveloped viruses, ofviral nuclear localization signals, of receptor-ligands, of celladhesion signals, of cell-targeting signals or of internalization- orendocytosis-triggering signals.

Examples of transfection enhancing agents include, but are not limitedto, insulin, a transferrin, epidermal growth factor, fibroblast growthfactor, a cell targeting antibody, a lactoferrin, a fibronectin, anadenovirus penton base, Knob, a hexon protein, a vesicular stomatitisvirus glycoprotein, a Semliki Forest Virus core protein, a influenzahemagglutinin, a hepatitis B core protein, an HIV Tat protein, a herpessimplex virus VP22 protein, a histone protein, a arginine rich cellpermeability protein, a high mobility group protein, and invasinprotein, and internalin protein, an endotoxin, a diptheria toxin, ashigella toxin, a melittin, a magainin, a gramicidin, a cecrophin, adefensin, a protegrin, a tachyplesin, a thionin, a indolicidin, abactenecin, a drosomycin, an apidaecin, a cathelicidin, abacteriacidal-permability-increasing protein, a nisin, a buforin, andfragments thereof.

Any proteins or peptides (or fragments or portions thereof) of theinvention may be used in accordance with this invention, either singlyor in combination with other proteins or peptides. In a preferredaspect, two or more, three or more, four or more, five or more, six ormore, etc. proteins and/or peptides are used in the invention.Additionally, such single or multiple proteins and/or peptides may beused in combination with one or more, two or more, three or more, fouror more, five or more, six or more, etc. transfection agents. In anotherpreferred aspect, at least two peptides and/or proteins are used incombination with a transfection agent, preferably at least twotransfection agents such as lipids, and/or polycations such asdendrimers or PEI.

Preparation and Use of Complexes Containing Fusogenic Peptides

The methods of the present invention involve contacting any cell,preferably a eukaryotic cell, with a transfection complex comprising atleast a fusogenic peptide, a transfection agent and a nucleic acid asdescribed above. The complex optionally may also contain one or moreadditional peptides or proteins, such as a fusogenic,membrane-permeabilizing, transport or traffickingsub-cellular-localization, or receptor-ligand peptide or protein. Theseadditional peptides or proteins optionally may be conjugated to anucleic acid-binding group, or optionally conjugated to the transfectionagent (lipid or polycationic polymer) where the peptide or protein ormodified peptide or protein is non-covalently associated with thenucleic acid. Without being bound by any theory, applicants believe thatthe complexes of the present invention are lipid aggregates thattypically contain liposomal structures, although the precise nature ofthese structures is not presently known. Accordingly, in certainillustrative examples, complexes of the present invention are liposomalcomplexes. The entire complex, or a portion of the complex, such as alipid portion, for example a lipid of Formula I, can be formulated intoliposomes, for example using the method of reverse evaporation, which iswell known in the art. Alternatively the lipid portion of the complex orthe entire complex, can be formulated by other well known methods forliposome formation such as sonication or microfluidization. Theseliposome formulations are effective for transfecting DNA into culturedcells.

In one embodiment, a complex containing the fusogenic peptide- orprotein of the invention and the nucleic acid (where the fusogenicpeptide or protein can be conjugated to a nucleic-acid binding group) isfirst formed and then combined with a cationic lipid, such as a lipid ofFormula I, for transfection. In a related embodiment, a peptide- orprotein-lipid conjugate is combined optionally with other lipids,including any appropriate cationic lipid, and then combined with nucleicacid for transfection. In another related embodiment, a nucleicacid-lipid complex is formed and then combined with a fusogenic peptideor protein for transfection. As discussed above, the lipid-containingcomplexes of any of these embodiments can be liposomal or non-liposomalformulations. Furthermore, any of the complexes formed in theseembodiments can be stored, for example, for 5 minutes to 1 year, or for15 minutes to 6 months, or for 1 hour to 3 months, before transfectingcells. In the case of a peptide or protein-lipid conjugate, such aconjugate can be stored for example, for 5 minutes to 1 year, or for 15minutes to 6 months, or for 1 hour to 3 months, before combining withnucleic acid.

In another embodiment, a complex containing the fusogenic peptide orprotein and the nucleic acid (where the fusogenic peptide or protein canbe conjugated to a nucleic-acid binding group) is formed and thencombined with a polycationic polymer for transfection. In a relatedembodiment, a peptide-polycationic polymer conjugate is combinedoptionally with another polycationic polymer and then combined withnucleic acid for transfection. In another related embodiment, a nucleicacid-polycationic polymer complex is formed and then combined with apeptide or protein for transfection. A polycationic polymer and/orpeptide-conjugated polycationic polymer can be combined with cationiclipids and cationic lipid composition to obtain improved nucleic acidtransfection compositions. In accordance with the invention, multiplepeptides and/or proteins may be added to accomplish transfection.

Transfection compositions of this invention comprising peptide- orprotein-lipid conjugates and nucleic acids can further include othernon-peptide or non-protein agents that are known to further enhancetransfection.

Transfection compositions of this invention comprising peptide- orprotein-polycationic polymer conjugates and nucleic acid can furtherinclude other non-peptide agents that are known to further enhancepolycationic polymer transfection, for example polycationic polymertransfection can be enhanced by addition of DEAE-dextran and/orchloroquine.

In one specific embodiment, the fusogenic peptide or protein,advantageously containing a polycationic sequence of amino acids, isfirst bound to a nucleic acid to be introduced into a cell. The peptide-or protein-nucleic acid complexes are then admixed with a transfectionagent (or mixture of agents) and the resulting mixture is employed totransfect cells. Preferred transfection agents are cationic lipidcompositions, such as those containing a lipid of Formula (I),particularly monovalent and polyvalent cationic lipid compositions, moreparticularly cationic lipid compositions composed of a 1:1 to 4:1mixtures of 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine andDOPE and a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine and cholesterolas well as a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine and DOPE anda 1:1 to 4:1 mixture of1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine andcholesterol; cationic lipid compositions composed of a 1:1 to 4:1mixtures of1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl]piperazine andDOPE and a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl]piperazine andcholesterol as well as a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine andDOPE and a 1:1 to 4:1 mixture of1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine andcholesterol; cationic lipid compositions composed of a 1:1 to 4:1mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine andDOPE and a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine andcholesterol as well as a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]piperazineand DOPE and a 1:1 to 4:1 mixture of1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]piperazineand cholesterol.

In a second specific transfection method, a transfection-enhancingpeptide or protein is conjugated to a nucleic acid-binding group, forexample a polyamine and more particularly a spermine, to produce amodified peptide or protein which is then bound to the nucleic acid tobe introduced into the cell. The modified peptide-nucleic acid complexis then admixed with a transfection agent (or mixture thereof) and theresulting mixture is employed to transfect cells. In particular, thepeptide or protein is covalently conjugated to a spermine, thespermine-modified peptide or protein is complexed with nucleic acid andadmixed with a cationic lipid. Preferred transfection agents arecationic lipid compositions, particularly monovalent and polyvalentcationic lipid compositions, more particularly cationic lipidcompositions composed of a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine and DOPE and a1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine and cholesterolas well as a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine and DOPE anda 1:1 to 4:1 mixture of1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine andcholesterol; cationic lipid compositions composed of a 1:1 to 4:1mixtures of1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl]piperazine andDOPE and a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl]piperazine andcholesterol as well as a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine andDOPE and a 1:1 to 4:1 mixture of1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine andcholesterol; cationic lipid compositions composed of a 1:1 to 4:1mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine andDOPE and a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)2-hydroxy-propyl]piperazine andcholesterol as well as a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]piperazineand DOPE and a 1:1 to 4:1 mixture of1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]piperazineand cholesterol.

In a third specific embodiment, a mixture of one or moretransfection-enhancing peptides, proteins, or protein fragments,including fusogenic peptides or proteins, transport or traffickingpeptides or proteins, receptor-ligand peptides or proteins, or nuclearlocalization peptides or proteins and/or their modified analogs (e.g.,spermine modified peptides or proteins) or combinations thereof aremixed with amino acid sequences from fusogenic proteins of non envelopedand complexed with nucleic acid to be introduced into a cell. Thepeptide-nucleic acid complexes are then admixed with transfection agentand the resulting mixture is employed to transfect cells. In certainembodiments, the mixture of the transfection enhancing peptide, protein,or protein fragment is stored before it is complexed with nucleic acid.

In another specific embodiment, a component of a transfection agent(lipids, cationic lipids, dendrimers, or PEI) is covalently conjugatedto selected peptides, proteins, or protein fragments directly or via alinking or spacer group. Of particular interest in this embodiment arepeptides or proteins that are fusogenic proteins from non-envelopedviruses.

Exemplary Uses of the Complexes Containing Fusogenic Peptides ofNon-Enveloped Viruses

The complexes and methods of the present invention, especially thoseinvolving transfection compositions that include complexes providedherein, can be used for in vitro and in vivo transfection of cells,particularly of eukaryotic cells, and more particularly to transfectionof higher eukaryotic cells, including animal cells. The methods of thisinvention can be used to generate transfected cells which express usefulgene products. The methods of this invention can also be employed as astep in the production of transgenic animals. The methods of thisinvention are useful as a step in any therapeutic method requiringintroduction of nucleic acids into cells including methods of genetherapy and viral inhibition and for introduction of antisense orantigene nucleic acids, ribozymes, RNA regulatory sequences, siRNA,RNAi, Stealth™ RNAi (Invitrogen Corporation, Carlsbad Calif.) or relatedinhibitory or regulatory nucleic acids into cells. In particular, thesemethods are useful in cancer treatment, in in vivo and ex vivo genetherapy, and in diagnostic methods.

The transfection compositions and methods of this invention comprisingpeptides, proteins, peptide or protein fragments or modified peptides ormodified proteins, can also be employed as research agents in anytransfection of eukaryotic cells done for research purposes.

Accordingly, provided herein is a method of introducing a macromoleculeinto a cell, that includes forming a transfection composition thatincludes a nucleic acid and a complex comprising a transfection agentand a fusion agent, wherein the fusion agent includes a fusion promotingamino acid sequence derived from a fusion protein of a non-envelopedvirus; and contacting a eukaryotic cell with the transfectioncomposition. Provided in the Examples section herein are illustrativeprotocols for using compositions of the present invention to transfecteukaryotic cells. As disclosed herein, the fusion agent in illustrativeexamples is a fusion peptide derived from a FAST protein of a Reovirus,advantageously a fusion peptide that is between 5 and 50 amino acids inlength where at least 10 contiguous amino acids of the fusion peptideare at least 75, 80, 85, 90, 95 or 100% identical to a hydrophobicregion of a Reovirus FAST protein, and a polycationic peptide sequence.

As illustrated in me Examples section below, volumes and concentrationsof nucleic acid or other macromolecule, volume and concentration of thetransfection complexes provided herein, volumes and compositions ofdiluents, and volume and concentration of cells, can be determined usingstandard experimental approaches for such optimization and titration,including, for example, methods that utilize cytotoxicity assays and/ormethods that employ transfection using nucleic acid expression vectorsthat express reporter genes, such as beta galactosidase, luciferase,and/or fluorescent proteins. Furthermore, cell densities can beoptimized using standard methods, and cell densities for transfectionsusing the transfection complexes provided herein can range, for example,from high density >75% to low density <50%

Exemplary diluents for complex formation, for example, includereduced-serum, or serum-free media, such as D-MEM and RPMI 1640 andOptiPro™, Opti-MEM® (Invitrogen Corporation). Incubation times forforming complexes can be determined using routine methods, althoughtypical incubation times are between 5 and 240 minutes. In addition, itwill be understood that media for culturing of cells before and aftertransfection can be chosen based on the cell line to be transfected andbased on the particular application of the method. For example, for theproduction of proteins in suspension cells, in illustrative embodiments,reduced serum, or advantageously serum-free, medium can be used. Incertain illustrative embodiments, animal origin-free medium is employed,such as, but not limited to, 293 Expression Medium (InvitrogenCorporation) and CD-CHO Medium (Invitrogen Corporation). In certainaspects depending on the cell type to be transfected, antibiotics can beexcluded from post-transfection media. Incubation times forpost-transfection culturing of cells varies depending on the cell typeand the desired outcome of the transfection, but typically ranges from 2hours to 7 days. For large-scale protein production, cells can beincubated, as a non-limiting example, for between 1 day and 7 days.

It will be understood that a wide range of concentrations oftransfection agent and a fusion agent can be used in the complexes,compositions and methods provided herein. For example, in anillustrative non-limiting example of a composition that includes acomplex of a cationic lipid and a fusogenic peptide, the totalexemplary, non-limiting combined concentration of cationic lipid andfusogenic peptide in the composition can be between 1 mg/ml and 4 mg/ml.The range of peptide added to the lipid at 1 mg/ml can between 100 μgmland 3 mg/ml. the ratio of the cationic lipid to helper lipid can between0.5/1.0 (molar) and pure compound.

Cells that can be transfected according to the present inventioninclude, for example, virtually any eukaryotic cell including primarycells, cells in culture, and cells in cultured tissue. The cells can beattached cells or cells in suspensions. In certain illustrative aspects,the cells are suspension CHO-S cells and suspension 293-F cells.Suspension cell cultures are particularly well-suited for proteinproduction methods provided herein. Other cells that can be transfectedusing the agents and methods of the invention include, but are notlimited to, 293, such as GripTite 293 MSR (Invitrogen Corporation), CHO,Cos7, NIH3T3, Hela, primary fibroblast, A549, Be2C, SW480, Caco2,primary neurons. Jurkat, C6, THP1, IMR90, HeLa, ChoK1, GT293, MCF7,HT1080, LnCap, HepG2, PC12, SKBR3, and K562 cells.

In certain embodiments provided herein, a transfection enhancing agentis included in the complex that is used to transfect cells. For examplethe transfection enhancing agent can be a nuclear localization peptide.In one example, the transfection enhancing agent is the PLUS™ Reagent(Invitrogen Corporation). It has been shown that the addition of PLUS™reagent enhances protein expression when used together with transfectioncompositions as provided herein. Expression was enhanced in NIH3T3,Jurkat, C6, Cos7 THP1, IMR90, LnCap, HepG2, PC12 and K562 cells.Cytotoxicity was not affected by the use of the PLUS™ Reagent.

In another embodiment, provided herein is a method for producing aprotein comprising, transfecting a cell with a nucleic acid moleculeencoding the protein, incubating the cell to produce the protein, andcollecting the protein, wherein the transfecting is performed bycontacting the cell with a transfection composition of the presentinvention. The composition for transfecting the cell can be anycompositions as provided herein. Exemplary compositions include thenucleic acid molecule encoding the protein of interest, a fusion agent,and typically a transfection agent, where the fusion agent includes afusion-promoting amino acid sequence derived from a fusion protein of anon-enveloped virus, such as a reovirus protein.

In illustrative embodiments the encoded protein is an antibody molecule,or an antigen binding fragment or derivative portion thereof, forexample a single chain Fv fragment. In these embodiments, the method canfurther include isolating the protein, for example, by using affinitypurification on an antibody-binding column. In certain examples, nucleicacids encoding both chains of an antibody are transfected into cellsusing a transfection composition provided herein.

It will be understood that the nucleic acid encoding the protein can bean expression vector. The expression vector typically has a promoteroperatively linked to one or more nucleic acid sequences encoding one ormore protein chains. Where the protein produced is a pharmaceuticalproduct, the protein can be formulated accordingly, for example in anappropriate choice of physiologic medium.

The transfection composition provided herein can also be used tointroduce peptides and proteins and the like into cells using methodsthat are known in the art. Methods of using cationic lipids for peptideand protein delivery previously have been described. In addition, thetransfection compositions can be used to deliver nucleic acids, peptidesand proteins and the like into tissues in vivo. Methods of using lipidsfor delivering compounds to tissue in vivo previously have beendescribed. The transfection compositions can, with appropriate choice ofphysiologic medium, be employed in therapeutic and diagnosticapplications.

Cell Transfections Using Lipids of Formula (I)

The lipids of Formula (I) can be used alone for delivery ofmacromolecules into cells in vitro or in vivo. These lipidsare at leastas active, and in most cases more active, than cationic lipids thatcurrently are commercially available for delivery of macromolecules intocells. Lipids of the present invention, for example the HCL salt of1,4-Bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine, wereformulated without co-lipid or with the neutral lipids DOPE orcholesterol (129A-E and 129H in Example 11). As shown in FIGS. 6-9, thelipids of the invention have been used to transfect CHO, NIH3T3, HEK293and 293GT cells, and were shown to afford transfection efficiencies thatwere 2-4-fold better than the comparison lipids.

The macromolecules which can be delivered into cells include, but arenot limited to, nucleic acids. The nucleic acid can be any type ofnucleic acid that presently is known or that may be prepared oridentified in the future, provided that the nucleic acid is sufficientlynegatively charged to form a lipid aggregate, liposome, or liposome-likecomplex when admixed with any lipid of Formula (I). Nucleic acid, asused herein, refers to deoxyribonucleotides or ribonucleotides andmixtures and polymers thereof in single- or double-stranded form. Theterm encompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as a reference nucleic acid, and which are metabolized in amanner similar to a reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs). The nucleic acid may be in the form of anantisense molecule, for example a “gap-mer”containing an RNA-DNA-RNAstructure that activates RNAseH. The nucleic acid can be, for example,DNA or RNA, or RNA-DNA hybrid, and can be an oligonucleotide, plasmid,parts of a plasmid DNA, pre-condensed DNA, product of a polymerase chainreaction (PCR), vectors, expression cassettes, chimeric sequences,chromosomal DNA, or derivatives of these groups or other form of nucleicacid molecule. The nucleic acid may be a double-stranded RNA molecule ofthe type used for inhibiting gene expression by RNA interference. Thenucleic acid may be a short interfering double stranded RNA molecule(siRNA). The nucleic acid molecule can also be a Stealth™ RNAi molecule(Invitrogen Corporation, Carlsbad, Calif.).

Accordingly, provided herein is a method of introducing macromoleculesinto cells. An exemplary method includes forming a lipid-nucleic acidcomplex using a lipid of Formula (I) and a nucleic acid, as describedherein, and contacting cells, such as, by way of example only,eukaryotic cells, with such a complex. The lipid may be in the form of alipid-aggregates, including, but not limited to, liposomes. The lipid ofFormula (I) may be used with a fusogenic peptide from a non-envelopedvirus according to the present invention, to transfect cells. It will beunderstood that incubation times, mixing protocols, and other specificaspects of the methods of the invention can be optimized using methodsknown in the art.

Cells which can be transfected according to the such methods include,but are not limited to, virtually any eukaryotic cell including primarycells, cells in culture, a passaged cell culture or a cell line, andcells in cultured tissue. Suitable cells include human cell lines andanimal cell lines. The cell may be a fibroblast. The cells can beattached cells or cells in suspensions. In certain illustrative aspects,the cells are suspension CHO-S cells and suspension 293-F cells. Othercells that may be used include, without limitation, 293, 293-S, CHO,Cos, 3T3, Hela, primary fibroblasts, A549, Be2C, SW480, CHOK1, Griptite293, HepG2, Jurkat, LNCap, MCF-7, NIH-3T3, PC12, C6, Caco-2, COS-7,HL60, HT-1080, IMR-90, K-562, SK-BR3, PHP1, HUVEC, MJ90, NHFF, NDFF andprimary neurons.

In another embodiment is a method for producing a protein which includescontacting a cell with a lipid-nucleic acid complex as described above,wherein the nucleic acid encodes the protein. The cells are incubated toproduce the protein and the protein is collected. Cells which can beused for protein production are described above. In addition, anycomposition which includes a lipid of Formula (I) can be used fortransfection of cells. Such compositions are further discussed herein,and include, but are not limited to compositions comprising lipids ofFormula (I), a co-lipid and an optional transfection enhancing agentsuch as a fusogenic peptide or protein.

The lipid aggregates of the present invention form a complex when theycome in contact with macromolecules such as nucleic acids. The lipidsoptionally may be used in slight excess and, in such as case, may form acationic complex. Without being bound by any theory, it is thought thatcationic complexes are attracted to the cell membrane therebyfacilitating uptake by the cell. Such lipid aggregates includeliposomes, unilamellar vesicles, multilamellar vesicles, micelles andthe like, which can have particle sizes in the nanometer to micrometerrange. The structure of various types of lipid aggregates varies,depending on composition and method of forming the aggregate. Methods ofmaking lipid aggregates are known in the art, and include, but are notlimited to, reverse evaporation, sonication and microfluidization.

In another embodiment, provided herein is a method for producing aprotein comprising, transfecting a cell with a nucleic acid encoding theprotein, incubating the cell to produce the protein, and collecting theprotein, wherein the transfecting is performed by contacting the cellwith a composition comprising a lipid of formula I, optionally with afusogenic peptide of a non-enveloped virus. The composition fortransfecting the cell can be any of the compositions provided herein,including those that include other lipids and/or additional peptides andproteins.

The lipids of Formula (I) may also be used to introduce peptides andproteins and the like into cells using methods that are known in theart. Methods of using cationic lipids for peptide and protein deliverypreviously have been described.

In addition, the lipids may be used to deliver nucleic acids, peptidesand proteins and the like into tissues in vivo. Methods of using lipidsfor delivering compounds to tissue in vivo previously have beendescribed.

Cationic lipid compositions composed of1,4-bis[(3-(3-aminopropyl)-alkylamino)propyl)piperazine lipids andneutral lipids, including 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine and DOPE and a1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine and cholesterolas well as a 1:1 to 4:1 mixtures of1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine and DOPE anda 1:1 to 4:1 mixture of1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine andcholesterol were effective at transfecting various cell types withnucleic acids.

Compositions and/or Methods that Use Other Lipids and/or Peptides

In certain illustrative examples, the lipids of Formula (I) also can beused in compositions with other lipids and/or with additionaltransfection-enhancing agents to deliver macromolecules. Suchcompositions may contain a lipid of Formula (I) and a co-lipid which isneutral, positively charged (such as a cationic lipid) or negativelycharged. Such neutral lipids include, but are not limited to,diacylphosphatidylcholine, diacylphosphatidylethanolamine (DOPE),ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides anddiacylglycerols. for example. The cationic lipids include, but are notlimited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol);N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE); 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA);1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), DODAP, DODMA, andDMDMA. The cationic lipids may also include, but are not limited to,LipofectAmine™ 2000, LipofectAmine™, Lipofectin®, DMRIE-C, Fugene®,Fugene® HD, Transfectam®, Transfectin™, SilentFect™, and Effectene®. Theanionic lipids include, but are not limited to, phosphatidylglycerol,cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid,N-dodecanoyl phosphatidylethanolamines, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines,lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG),and other anionic modifying groups joined to neutral lipids.

Transfection enhancing agents which may be included in the compositionsdescribed above include, but are not limited to, transfection-enhancingpeptides or proteins that function to deliver the macromolecule tospecific sub-cellular locations such as the nucleus or other organelles,that function for cellular transport or trafficking, that are receptorligands, that comprise cell-adhesion signals, cell-targeting signals,cell-internalization signals or endocytosis signals. Other examplesinclude peptides or functional portions thereof that are enveloped ornon-enveloped viral proteins or derived from enveloped or non-envelopedviral proteins, that are enveloped or non-enveloped viral fusogenicproteins or derived from enveloped or non-enveloped viral fusogenicproteins, that contain viral nuclear localization signals, that arereceptor-ligands, that contain cell adhesion signals, cell-targetingsignals, and/or internalization- or endocytosis-triggering signals.Exemplary fusogenic peptides are the reovirus-derived peptides describedherein.

In certain embodiments provided herein, transfection enhancing agent canbe a nuclear localization peptide. In one example, the transfectionenhancing agent is the PLUS™ Reagent (Invitrogen Corporation). It hasbeen determined in initial experiments that the addition of PLUS™reagent enhances protein expression when used along with transfectioncompositions provided herein. In fact, expression was enhanced inNIH3T3, Jurkat, C6, Cos7 THP1, IMR90, LnCap, HepG2, PC12 and K562 cells.Cytotoxicity was not affected by the use of the PLUS™ Reagent.

In an illustrative example, the lipids of the present invention are usedin conjunction with Plus Reagent™ (available from InvitrogenCorporation, Carlsbad, Calif.), as provided in exemplary transfectionprotocols provided herein. Furthermore, the lipids can be formulated inliposomal or non-liposomal formulations, that can include a helperlipid, such as DOPE, along with a Prototypical Avian reovirus fusogenicpeptide, as described above, and used in conjunction with Plus Reagent™to deliver nucleic acids to cells.

Exemplary peptides or proteins that may be used in combination with thelipids of formula I include those derived from enveloped and nonenveloped viruses, bacteria, insulin, a transferrin, a epidermal growthfactor, a fibroblast growth factor, a cell targeting antibody, alactoferrin, a fibronectin, an adenovirus penton base, Knob, a hexonprotein, a vesicular stomatitis virus glycoprotein, a Semliki ForestVirus core protein, a influenza hemagglutinin, a hepatitis B coreprotein, an HIV Tat protein, a herpes simplex virus VP22 protein, a reovirus fusion protein or peptide, a histone protein, an arginine richcell permeability protein, a high mobility group protein, and invasinprotein, and internalin protein, an endotoxin, a diptheria toxin, ashigella toxin, a melittin, a magainin, a gramicidin, a cecrophin, adefensin, a protegrin, a tachyplesin, a thionin, a indolicidin, abactenecin, a drosomycin, an apidaecin, a cathelicidin, a adapatinprotein, a bacteriacidal-permability-increasing protein, a nisin, abuforin, and fragments thereof.

The novel lipids of Formula (I) may be formulated with one or morenucleic acids into liposomes or liposome-like vehicles in the presenceor absence of co-lipid such as, by way of example only,dioleylphosphatidyl ethanolamine (DOPE) or cholesterol. The lipids maybe formulated into liposomes, for example using the method of reverseevaporation, which is well known in the art. Alternatively the lipidsmay be formulated by other well known methods for liposome formationsuch as sonication, microfluidization etc. These liposome formulationsare effective for transfecting DNA into cultured cells.

In one method, a nucleic acid is contacted with a fusion agent and theresulting mixture is added to a mixture of a lipid of Formula (I) and aneutral lipid, where the fusion agent contains a fusion-promoting aminoacid sequence derived from a fusion protein of a non-enveloped virus, asdescribed in more detail above and in the Examples below.

In another method, a fusion agent is contacted with a lipid of Formula(I) followed by addition of a nucleic acid or protein capable ofaggregating the peptide- or protein-nucleic acid complex, where thefusion agent contains a fusion-promoting amino acid sequence derivedfrom a fusion protein of a non-enveloped virus, as described in moredetail above and in the Examples below.

In certain embodiments of the present invention methods involvecontacting any cell, preferably a eukaryotic cell, with a transfectioncomplex comprising at least a fusogenic peptide, a lipid of Formula (I)and a nucleic acid as described above. The complex optionally may alsocontain one or more additional peptides or proteins, such as afusogenic, membrane-permeabilizing, transport or traffickingsub-cellular-localization, or receptor-ligand peptide or protein. Theseadditional peptides or proteins optionally may be conjugated to anucleic acid-binding group, or optionally conjugated to a lipid ofFormula (I) where the peptide or protein or modified peptide or proteinis non-covalently associated with the nucleic acid. Without being boundby any theory, applicants believe that the complexes of the presentinvention are lipid aggregates that typically contain liposomalstructures, although the precise nature of these structures is notpresently known. Accordingly, in certain illustrative examples,complexes of the present invention are liposomal complexes. The entirecomplex, or a portion of the complex, such as a lipid portion, can beformulated into liposomes, for example using the method of reverseevaporation, which is well known in the art. Alternatively the lipidportion of the complex or the entire complex, can be formulated by otherwell known methods for liposome formation such as sonication ormicrofluidization. These liposome formulations are effective fortransfecting DNA into cultured cells.

In one embodiment, a complex containing the fusogenic peptide or proteinand the nucleic acid (where the fusogenic peptide or protein can beconjugated to a nucleic-acid binding group) is first formed and thencombined with a cationic lipid for transfection. In a relatedembodiment, a peptide- or protein-lipid conjugate is combined optionallywith other lipids, including any appropriate cationic lipid, and thencombined with nucleic acid for transfection. In another relatedembodiment, a nucleic acid-lipid complex is formed and then combinedwith a fusogenic peptide or protein for transfection. As discussedabove, the lipid-containing complexes of any of these embodiments can beliposomal or non-liposomal formulations.

The complexes and methods of the present invention, especially thoseinvolving transfection compositions that include complexes providedherein, can be used for in vitro and in vivo transfection of cells,particularly of eukaryotic cells, and more particularly to transfectionof higher eukaryotic cells, including animal cells. The methods of thisinvention can be used to generate transfected cells which express usefulgene products. For example, the methods can be used to producerecombinant antibody molecules, typically by expressing a recombinantlight chain molecule and a recombinant heavy chain molecule from one ormore expression vectors that are introduced into a cell, especially asuspension cell, using the complexes provided herein. The methods ofthis invention can also be employed as a step in the production oftransgenic animals. The methods of this invention are useful as a stepin any therapeutic method requiring introduction of nucleic acids intocells including methods of gene therapy and viral inhibition and forintroduction of antisense or antigene nucleic acids, ribozymes, RNAregulatory sequences, siRNA, RNAi, Stealth™ RNAi (InvitrogenCorporation, Carlsbad Calif.) or related inhibitory or regulatorynucleic acids into cells. In particular, these methods are useful incancer treatment, in in vivo and ex vivo gene therapy, and in diagnosticmethods.

The transfection compositions and methods of this invention comprisingpeptides, proteins, peptide or protein fragments or modified peptides ormodified proteins, can also be employed as research agents in anytransfection of eukaryotic cells done for research purposes.

Accordingly, provided herein is a method of introducing a macromoleculeinto a cell, that includes forming a transfection composition thatincludes a nucleic acid and a complex comprising a lipid of Formula (I)and a fusion agent, wherein the fusion agent includes a fusion promotingamino acid sequence derived from a fusion protein of a non-envelopedvirus; and contacting a eukaryotic cell with the transfectioncomposition. Provided in the Examples section herein are illustrativeprotocols for using compositions of the present invention to transfecteukaryotic cells. As disclosed herein, the fusion agent in illustrativeexamples is a fusion peptide derived from the FAST protein of aReovirus, most preferably a fusion peptide that is between 5 and 50amino acids in length wherein at least 10 contiguous amino acids of thefusion peptide are at least 75, 80, 85, 90, 95 or 100% identical to ahydrophobic region of a Reovirus FAST protein, and a polycationicpeptide sequence.

It will be understood that quantities, concentrations and volumes ofcomplexes, complex components, and nucleic acid or other macromolecules,incubation times, mixing protocols, and other specific aspects of themethods of the invention are known in the art or can be optimized and/oridentified using methods known in the art. As illustrated in theExamples section herein, volumes and concentrations of nucleic acid orother macromolecule, volume and concentration of the transfectioncomplexes provided herein, volumes and compositions of diluents, andvolume and concentration of cells, can be determined using standardexperimental approaches for such optimization and titration, including,for example, methods that utilize cytotoxicity assays and/or methodsthat employ transfection using nucleic acid expression vectors thatexpress reporter genes, such as beta galactosidase, luciferase, and/orfluorescent proteins. Furthermore, cell densities can be optimized usingstandard methods, and cell densities for transfections using thetransfection complexes provided herein can range, for example, from highdensity >75% to low density <50%

Exemplary diluents for complex formation, for example, include reducedserum, or serum-free media, such as D-MEM and RPMI 1640 and OptiPro™,Opti-MEM® (Invitrogen Corporation). Incubation times for formingcomplexes can be determined using routine methods, although typicalincubation times are between 5 and 240 minutes. In addition, it will beunderstood that media for cell culturing can be chosen based on the cellline to be transfected and based on the particular application of themethod. For example, for the production of proteins in suspension cells,in illustrative embodiments, reduced serum, and preferably serum-freemedium can be used. In certain illustrative embodiments, animal originalfree medium is employed, such as, but not limited to, 293 ExpressionMedium (Invitrogen Corporation) and CD-CHO Medium (InvitrogenCorporation). In certain aspects depending on the cell type to betransfected, antibiotics can be excluded from post-transfection media.Incubation times for post-transfection culturing of cells variesdepending, but typically ranges from 2 hours to 7 days. For large-scaleprotein production, cells can be incubated, as a non-limiting example,for between 1 day and 7 days.

It will be understood that a wide range of concentrations of lipids ofFormula (I), co-lipids and transfection enhancing agents can be used inthe complexes, compositions and methods provided herein. For example, inan illustrative non-limiting example of a composition provided hereinthat includes a complex of a lipid of Formula (I) and a fusogenicpeptide, the total exemplary, non-limiting combined concentration oflipid of Formula (I) and fusogenic peptide in the composition can bebetween 1 mg/ml and 4 mg/ml. The range of peptide added to the lima at 1mg/ml can be between 100 μgml and 3 mg/ml. the ratio of the helper lipidto cationic lipid can be between 0.25:1.0 (molar) and pure compound

Cells that can be transfected according to the present inventioninclude, for example, virtually any eukaryotic cell including primarycells, cells in culture, and cells in cultured tissue. The cells can beattached cells or cells in suspensions. In certain illustrative aspects,the cells are suspension CHO-S cells and suspension 293-F cells. Othercells that can be transfected using the agents and methods of theinvention include, but are not limited to, 293, such as GripTite 293 MSR(Invitrogen Corporation), CHO, Cos7, NIH3T3, Hela, primary fibroblast,A549, Be2C, SW480, Caco2, primary neurons. Jurkat, C6, THP1, IMR90,HeLa, ChoK1, GT293, MCF7, HT1080, LnCap, HepG2, PC12, SKBR3, and K562cells.

In another embodiment, provided herein is a method for producing aprotein comprising, transfecting a cell with a nucleic acid moleculeencoding the protein, incubating the cell to produce the protein, andcollecting the protein, wherein the transfecting is performed bycontacting the cell with a transfection composition of the presentinvention. The composition for transfecting the cell can be any of thecompositions provided herein. By way of example, such compositions caninclude the nucleic acid molecule encoding the protein, a fusion agent,and a lipid if Formula (I), wherein the fusion agent include a fusionpromoting amino acid sequence derived from a fusion protein of anon-enveloped virus, such as a reovirus protein.

Pharmaceutical Compositions

Transfection agents and transfection-enhancing agents of this inventioncan be provided in a variety of pharmaceutical compositions and dosageforms for therapeutic applications. For example, injectableformulations, intranasal formulations and formulations for intravenousand/or intralesional administration containing these complexes can beused therapy.

In general the pharmaceutical compositions of this invention shouldcontain sufficient transfection agent and any enhancing agents (peptide,protein, etc.) to provide for introduction of a sufficiently high enoughlevel of nucleic acid into the target cell or target tissue such thatthe nucleic acid has the desired therapeutic effect therein. The levelof nucleic acid in the target cell or tissue that will betherapeutically effective will depend on the efficiency of inhibition orother biological function and on the number of sites the nucleic acidmust affect.

The dosage of transfection compositions described herein administered toa patient will depend on a number of other factors including the methodand site of administration, patient age, weight and condition. Those ofordinary skill in the art can readily adjust dosages for a given type ofadministration, a given patient and for a given therapeutic application.

It will be appreciated by those of ordinary skill in the art that thetransfection composition should contain minimal amounts of inhibitorycomponents, such as serum or high salt levels, which may inhibitintroduction of nucleic acid into the cell, or otherwise interfere withtransfection or nucleic acid complexation. It will also be appreciatedthat any pharmaceutical or therapeutic compositions, dependent upon theparticular application, should contain minimal amounts of componentsthat might cause detrimental side-effects in a patient.

The transfection compositions described herein may be formulated intocompositions which include a pharmaeceutically active agent and apharmaceutically acceptable diluents, excipients or carriers therefor.Such compositions may be in unit dosage forms such as tablets, pills,capsules (including sustained-release or delayed-release formulations),powders, granules, elixirs, tinctures, syrups and emulsions, sterileparenteral solutions or suspensions, aerosol or liquid sprays, drops,ampoules, auto-injector devices or suppositories; for oral, parenteral(e.g., intravenous, intramuscular or subcutaneous), intranasal,sublingual or rectal administration, or for administration by inhalationor insufflation, and may be formulated in an appropriate manner and inaccordance with accepted practices such as those disclosed inRemington's Pharmaceutical Sciences, (Gennaro, ed., Mack Publishing Co.,Easton Pa., 1990, herein incorporated by reference).

Some examples of suitable carriers, excipients and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, methyl cellulose, methyl- and propyl-hydroxybenzoates, talc,magnesium stearate and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents or flavoring agents. Whenthe carrier serves as a diluent, it may be a solid, semi-solid or liquidmaterial which acts as a vehicle, excipient or medium for the activeingredient. In the case of injections, it is possible to preparesolutions or liposomes of one or more lipids of the present invention inpharmaceutically acceptable carriers such as an aqueous or nonaqueoussolvent. Examples of solvents which may be used are distilled water forinjection, physiological saline solution, Ringer's solution, plant oil,synthetic fatty acid glycerides, higher fatty acid esters, propyleneglycol, and the like.

Reagent Kits

Components of the transfection compositions of this invention can beprovided in a reagent kit. The kit may contain a transfection agent andan amino acid sequence from a fusogenic protein of a non envelopedvirus. This kit can also include a transfection enhancing agent such asa transfection-enhancing peptide, protein or fragment thereof or atransfection enhancing compound. The transfection agent, the amino acidsequence, typically a fusogenic peptide, and the transfection enhancingagent, when present, can each be included as a mixture (i.e. in a singlecontainer, typically a tube and/or vial), or can be included as separateportions (i.e. in separate containers, for example separate vials and/ortubes). The kits of the present invention, as will be understood,typically include vessels, such as vials and/or tubes, that are packagedtogether, for example in a cardboard box. The kits can be shipped from asupplier to a customer. For example, in one example provided herein is akit that includes a vial that includes a liposomal formulation thatincludes a transfection agent and a transfection enhancing peptide. Thekit can also include, for example, a separate vessel that includes atransfection enhancing agent, such as a transfection enhancing peptide,for example Plus Reagent™ (Invitrogen Corp., Carlsbad, Calif.). The kitcan also include in separate containers, cells, cell culture medium, anda reporter nucleic acid sequence, such as a plasmid that expresses areporter gene. In certain examples, the culture medium can bereduced-serum medium and/or protein expression medium.

In one embodiment, a kit comprises individual portions of, or a mixtureof, cationic lipid, such as a lipid of Formula I, and peptide, proteinor fragment thereof or modified peptide, protein or fragment thereof. Inanother embodiment, a kit comprises individual portions of or a mixtureof, polycationic polymers and peptide, protein or fragments thereof ormodified peptide, protein or fragments thereof. Cationic lipidtransfection kits can optionally include neutral lipid as well as othertransfection-enhancing agents or other additives, and the relativeamounts of components in the kit may be adjusted to facilitatepreparation of transfection compositions. Kit components can includeappropriate medium or solvents for other kit components.

Cationic lipid transfection kits comprising a monocationic orpolycationic lipid composition, such as a lipid of Formula I, andfurther including a neutral lipid and a modified peptide or protein arepreferred. Dendrimer transfection kits can optionally include othertransfection enhancing agents, such as DEAE-dextran and/or chloroquine,as well as other additives and the relative amounts of components in thekit may be adjusted to facilitate preparation of transfectioncompositions. Kits provided by this invention include those comprisingan individual portion of a polycationic lipid composition comprisingDOSPA and DOPE or a monocationic lipid composition comprising DOTMA andDOPE and a portion of modified peptide, particularly a spermine-modifiedpeptide. Kits provided by this invention include those comprising anindividual portion of a polycationic polymer and a portion of aspermine-modified peptide.

In related embodiments, kits of this invention can comprise a peptide-or protein-lipid conjugate or a peptide- or protein-polycationic polymerconjugate in combination with non-conjugated lipids, non-conjugatedpolycationic polymer and other agents to facilitate transfection.

Kits of this invention can include those useful in diagnostic methods,e.g., diagnostic kits which in addition to transfection agent andtransfection-enhancing agents (e.g., proteins, peptides, and fragmentsand modifications of peptides and proteins) can contain a diagnosticnucleic acid. A diagnostic nucleic acid is a general term for anynucleic acid which can be employed to detect the presence of anothersubstance (most generally an analyte) in a cell. For example, whentransfected into a cell a diagnostic nucleic acid may increase ordecrease expression of a gene therein in response to the presence ofanother substance in the cell (e.g., a protein, small molecule, steroid,hormone, or another nucleic acid). Diagnostic nucleic acids also includethose nucleic acids that carry some label or otherwise detectable markerto a particular target cell or target tissue for detection of the targetcell or tissue or for detection of a substance in the target cell ortissue.

Nucleic acids that can be transfected by the methods of this inventioninclude DNA and RNA of any size from any source comprising natural basesor non-natural bases, and include those encoding and capable ofexpressing therapeutic or otherwise useful proteins in cells, thosewhich inhibit undesired expression of nucleic acids in cells, thosewhich inhibit undesired enzymatic activity or activate desired enzymes,those which catalyze reactions (ribozymes), and those which function indiagnostic assays (e.g., diagnostic nucleic acids). Therapeutic nucleicacids include those nucleic acids that encode or can expresstherapeutically useful proteins, peptides or polypeptides in cells,those which inhibit undesired expression of nucleic acids in cells,those which inhibit undesired enzymatic activity or activate desiredenzymes in cells.

The compositions and methods provided herein can also be readily adaptedin view of the disclosure herein to introduce biologically-activemacromolecules other than nucleic acids including, among others,polyamines, polyamine acids, polypeptides and proteins into eukaryoticcells. Other materials useful, for example as therapeutic agents,diagnostic materials, research reagents, which can be bound to thepeptides and modified peptides and introduced into eukaryotic cells bythe methods of this invention.

The lipids of Formula I can be used as the cationic lipid(s) of the kitsdescribed above, and may independently be provided in a reagent kit. Ingeneral, the kit contains a lipid of Formula (I) in a suitablecontainer. The lipid may be. for example, in a solution of an organicsolvent, such as ethanol, in a buffer, or in a solvent/buffer mixture Inaddition, the kit may include, but is not limited to, a lipid of Formula(I), and an amino acid sequence from a fusogenic protein of a nonenveloped virus in a suitable solvent or buffer.

In one embodiment, a kit may comprise individual portions of, or amixture of, lipids of Formula (I) and peptide, protein or fragmentthereof or modified peptide, protein or fragment thereof. Kits whichinclude lipids of Formula (I) can optionally include neutral lipid aswell as other transfection-enhancing agents or other additives, and therelative amounts of components in the kit may be adjusted to facilitatepreparation of transfection compositions. Kit components can includeappropriate medium or solvents for other kit components.

Kits which include lipids of Formula (I), a neutral lipid and a modifiedpeptide or protein are preferred. Kits provided by this inventioninclude those composition comprising an individual portion of a lipid ofFormula (I), DOPE and a portion of modified peptide, particularly aspermine-modified peptide. Kits provided by this invention include thosecomprising an individual portion of a lipid of Formula (1), and aportion of a modified peptide containing a stretch of basic amino acidssuch lysine, ornithine, or arginine.

Methods for Selling

Also provided is a method for selling a fusogenic peptide, lipid,transfection complex, transfection composition, and/or kit providedherein, comprising presenting to a customer an identifier thatidentifies the fusogenic peptide, lipid, complex and/or transfectioncomposition, and/or a kit provided herein, and providing access to thecustomer to a purchase function for purchasing the fusogenic peptide,lipid, transfection complex, transfection composition, and/or kitprovided herein using the identifier. The identifier is typicallypresented to the customer as part of an ordering system. The orderingsystem can include an input function for identifying a desired product,and a purchasing function for purchasing a desired product that isidentified. The ordering system is typically under the direct orindirect control of a provider. A customer as used herein, refers to anyindividual, institution, corporation, university, or organizationseeking to obtain biological research products and services. A provideras used herein, refers to any individual, institution, corporation,university, or organization seeking to provide biological researchproducts and services.

The present invention also provides a method for selling a fusogenicpeptide, lipid, transfection complex, transfection composition, and/orkit provided herein, comprising: presenting to a customer an inputfunction of a telephonic ordering system, and/or presenting to acustomer a data entry field or selectable list of entries as part of acomputer system,

wherein the fusogenic peptide, lipid, transfection complex, transfectioncomposition and/or kit is identified using the input function. Where theinput function is part of a computer system, such as displayed on one ormore pages of an Internet site, the customer is typically presented withan on-line purchasing function, such as an online shopping cart, whereinthe purchasing function is used by the customer to purchase theidentified fusogenic peptide, lipid, transfection complex, transfectioncomposition, and/or kit. In one aspect, a plurality of identifiers areprovided to a customer, each identifying a different fusogenic peptide,lipid, complex and/or transfection composition, and/or a kit providedherein, or a different volume or weight of the fusogenic peptide, lipid,complex and/or transfection composition, and/or a kit provided herein.The method may further comprise activating the purchasing function topurchase the lipid, transfection complex, transfection composition,and/or kit provided herein. The method may still further compriseshipping the purchased fusogenic peptide, lipid, transfection complex,transfection composition, and/or kit provided herein to the customer.The fusogenic peptide, lipid, transfection complex, transfectioncomposition, and/or kit can be shipped by a provider to the customer.The provider typically controls the input function, and can control theweb site accessed to access the input function to purchase a fusogenicpeptide, lipid, complex and/or transfection composition, and/or a kitprovided herein.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1 “After” Transfection Protocol where Peptide is Addedto DNA/Lipid Complex

Transfection of CHO-K1, NIH3T3, A549, Cos-7 and BE(2)C withβ-galactosidase reporter plasmid pCMV•SPORT-β-gal was carried out asfollows:

Cells were plated in a 96-well plates with 100 μl of media containing10% fetal calf serum the day before transfection such that a desiredconfluency (70%-95%) was achieved.

The following day a transfection agent that includes a liposomalcomposition of the lipid DMTS (Dimyrstyl-tetrahydroxy-spermine) and DOPE(2:1 DMTS:DOPE) and DNA were mixed in Opti-MEM to form DNA/lipidcomplexes. Complexes were formed by adding various amounts of lipids(0.1 to 0.35 μl) to 100 μl of Opti-MEM. DNA (100 ng) was added to 100 μlOpti-MEM. The DNA and lipid solutions were then mixed to form DNA lipidcomplexes. The complexes were incubated for at least 15 minutes afterwhich added to the DNA lipid complexes, was 20 μl of various amounts ofa peptide having the sequence of a Prototypical Avian reovirus fusogenicpeptide provided herein, as follows:

Arg-Met-Pro-Pro-Gly-Ser-Cys-Asn-Gly-Ala-Thr-Ala-Val-Phe-Gly-Asn-Val-His-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys,

and incubated for 15 minutes to form a DNA/lipid/peptide complex. Afterincubation of 30 minutes, 20 μl of DNA/lipid/peptide complex was addeddirectly to the cells in 10% serum. Cells were incubated for anadditional 24 hours to allow expression of the plasmid. Medium wasremoved and the cells were lysed in 100-200 μl of lysis buffer. Thelysates (20 μl) were assayed for β-gal activity using the enzymaticsubstrate ONPG. Total activity was determined by reading the OD at 405using Bio-Rad Benchmark Microplate Spectrophotometer.

Example 2 “Before” Transfection Protocol where Peptide is Mixed with DNAand Added to Lipid to Form Complex

Transfection of CHO-K1, NIH3T3, A549, Cos-7 and BE(2)C withβ-galactosidase reporter plasmid pCMV•SPORT-β-gal was carried out asfollows:

Cells were plated in 96-well plates with 100 μl of media containing 10%fetal calf serum the day before transfection such that a desiredconfluency (70%-95%) was achieved. The following day a transfectionagent that includes a liposomal composition of the lipid DMTS(Dimyrstyl-tetrahydroxy-spermine) and DOPE (2:1 DMTS:DOPE) andDNA/peptide were mixed in Opti-MEM to form DNA/lipid/peptide complexes.The peptide and DNA were mixed for 15 minutes and the mixed with lipidfor an addition 15 minutes Complexes were formed by adding variousamounts of lipids (0.1 to 0.35 μl) to 100 μl of Opti-MEM. DNA (100 ng)was added to 100 μl Opti-MEM then various amounts of peptide having thesequence of Prototypical Avian reovirus fusogenic peptides providedherein, as follows:

Arg-Met-Pro-Pro-Gly-Ser-Cys-Asn-Gly-Ala-Thr-Ala-Val-Phe-Gly-Asn-Val-His-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys

were added to the DNA mixture and incubated for 15 minutes. TheDNA/peptide and lipids solutions were then mixed to form DNA lipidcomplexes. The complexes were incubated for an additional 15 minutes.After incubation, 20 μl of complexes were added directly to the cells in10% serum. Cells were incubated for an additional 24 hours to allowexpression of the plasmid. Medium was removed and the cells were lysedin 100-200 μl of lysis buffer. The lysates (20 μl) were assayed forβ-gal activity using the enzymatic substrate ONPG. Total activity wasdetermined by reading the OD at 405 using Bio-Rad Benchmark MicroplateSpectrophotometer.

In all examples, as shown in FIG. 1-5, the addition of the peptide tothe DNA and then adding cationic lipid or adding peptide to theDNA/lipid complex gave enhancement of transfection and reduced theamount of lipid required to enhance transfection. For optimal results,the choice of whether to use the before or after transfection methodwill depend on the nature of the cell line that is being transfected.Other lipids such as DMRIE-C, LIPOFECTAMINE™, and Fugene6 were found tobe enhanced by addition of the peptide using the before method

Example 3 Exemplary Transfection Protocol for Cells in Suspension

Transfection of CHO-S and HEK293 in suspension with β-galactosidasereporter plasmid pCMV•SPORT-β-gal is carried out as described below.Prior to transfection the cells are cultivated in suspension in ahumidified 37° C. and 8% CO₂ on an orbital shaker. Antibiotics are notadded to the media as this may cause cell death. In addition clumpingcan lower transfection efficiency therefore the cells are sufficientlyagitated at regular intervals to avoid clumping and anti-clumping agentsare not added during culturing and prior to transfection. However,anti-clumping agents are optionally used post-transfection.

-   -   A) For routine culturing of HEK293 cells, shake at 135-155 rpm        keeping the cell densities between 0.1 and 2×10⁶ cells/mL of        culture. A cell density above 2×10⁶ cells/mL will result in a        loss of transfection efficiency.    -   B) For routine culturing of CHO-S cells, shake at 120-135 rpm        keeping the cell densities between 0.05 and 1.5×10⁶ cells/mL of        culture. A cell density above 1.5×10⁶ cells/mL will result in a        loss of transfection efficiency. The media is supplemented with        L-glutamine to a final concentration 8 mM.

Approximately 24 hours before transfection, CHO-S cells (at 5-6×10⁵cells/ml) or HEK293 cells (at 6-7×10⁵ cells/mL), are placed in a flaskon an orbital shaker platform rotating at 135-155 rpm at (HEK293 cells)or at 120-135 rpm at (CHO-S cells) at 37° C. and 8% CO₂ and allowed tocontinue to cultivate. The following day, the cells are diluted to about1×10⁶ cells/ml and then 30 mL of the diluted cells are placed into a 125mL shake flask. Then in a tube 30 μg of plasmid DNA is mixed withOpti-Pro™ SFM medium to a total volume of 0.6 mL, and in a separate tube30 μL of a transfection composition provided herein at 2 mg/ml totalconcentration of fusogenic peptide of Example 1, neutral lipid, andcationic lipid is mixed with Opti-Pro™ SFM medium to a total volume of0.6 mL. The transfection composition solution is then added to thediluted DNA solution giving a total volume of 1.2 mL. This mixture isthen incubated 10 minutes at room temperature to allow theDNA-lipid-fusogenic complex to form. The solution (1.2 mL) ofDNA-lipid-fusogenic peptide complex is then slowly added to the 125 mLflask containing the cells while gently swirling the flask.

The transfected cell culture is incubated at 37° C. and 8% CO₂ on anorbital shaker platform rotating at 135-155 rpm for an additional 24hours to allow expression of the plasmid. Medium is removed and thecells are lysed in 100-200 μl of lysis buffer. The lysates (20 μl) areassayed for β-gal activity using the enzymatic substrate ONPG. Totalactivity is determined by reading the OD at 405 nm using a Bio-RadBenchmark Microplate Spectrophotometer. Protein expression can bedetected within 4 to 8 hours, with maximal protein yield usually between1-7 days post transfection. This method was successfully employed totransfect cells and express proteins.

Optimizing Protein Expression

To optimize protein expression a time course is obtained between days 1and 7 post transfection and the peak of protein production is obtainedand cell viability is monitored. To assess transfection efficiency viaexpression of a GFP-type fluorescent protein the culture is monitoredstarting 24 hours post transfection. For secreted IgG protein productionthe peak yields are at 5-7 days post transfection.

Scaling Up or DownTransfections

For different culture volumes the following parameters are used:

Culture Culture Rotation Dilution DNA Ttransfection Complex Volume FlaskSpeed Volume quantity composition time Cells (mL) (mL) (rpm) (mL) (μg)(μL) (min.) CHO—S 30 125 135  1.2 30 30 10-20 (2 × 0.6 mL) or HEK293 30125 135  1.2 30 30 10-20 (2 × 0.6 mL) CHO—S 200 500 135  8 200 200 10-20(2 × 4 mL) or HEK293 200 500 135  8 200 200 10-20 (2 × 4 mL CHO—S 4001000 135 16 400 400 10-20 (2 × 8 mL) or HEK293 400 1000 135 16 400 40010-20 (2 × 8 mL) CHO—S 1000 3000 70 40 1.25 1.25 20 (2 × 20 mL) orHEK293 1000 3000 ≦135 40 1.0 1.0 10-20 (2 × 20 mL)Note: The lipid is tested for absence of microbial contamination usingblood agar plates, Sabraud dextrose agar plates and fluid thioglycolatemedium and functionally by transfection with a reporter plasmid.

Example 4 Single Tube Protocol

The following protocol is used to transfect DNA (β-galactosidasereporter plasmid pCMV•SPORT-β-gal) into mammalian cells in a 24-wellformat. The amounts used are on a per well basis.

Adherent Cells

One day before transfection, plate cells in 500 μL of growth medium sothat the cells are 50-80% confluent at the time of transfection.

Suspension Cells

Just prior to preparation of the complexes, 200,000-500,000 cells areplated in 500 μL of growth medium.

Transfection

For each transfection sample the complexes are prepared as follows.

500 ng of plasmid DNA is diluted in 100 μL Opti-MEM® I Reduced SerumMedium without serum and is gently mixed. (For optional transfectionswith Plus™ Reagent (Invitrogen Inc., Carlsbad Calif.), the Plus™ Reagentis mixed gently and 0.5 μL is added directly to the diluted DNA, andincubated for 5 minutes at room temperature.) Then 1.25 μL of atransfection composition provided herein that includes a fusogenicpeptide, a cationic lipid, and a helper lipid is added to the dilutedDNA solution (with or without Plus™ Reagent) and the mixture is gentlymixed and is incubated for 30 minutes at room temperature to form thecomplexes. Then approximately 100 μL of the complex is added directly toa well containing the cells and the plate is gently rocked back andforth. Cells are incubated at 37° C. in a CO₂ incubator for anadditional 24 hours to allow expression of the plasmid. Medium isoptionally changed after 4-6 hours.

Medium is removed and the cells are lysed in 100-200 μl of lysis buffer.The lysates (20 μl) are assayed for β-gal activity using the enzymaticsubstrate ONPG. Total activity was determined by reading the OD at 405nm using a Bio-Rad Benchmark Microplate Spectrophotometer.

Optimizing Transfections

To obtain the highest transfection performance, the amounts of DNA andlipid are varied as follows for a 24-well format (for other formats, theamounts are adjusted accordingly.)

Transfection Optional Plus ™ DNA compostion Reagent Cells (ng) (μL) (μL)Sensitive cells (HeLa, 250 0.375-1.25 1.25-0.5 HT1080) Most cell lines500 0.75-3.0 0.25-1.0 700 1.125-4.5  0.375-1.5  Suspension cells 1000 1.5-5.0  0.5-2.0 And Robust cells (e.g. Jurkat, THP1 and HL60)

Generating Stable Cells Lines

Cells are passed at 1:10 (or higher dilution) into fresh medium 1 dayafter transfection. Selective medium is optionally added the next day.

Scaling Up or DownTransfections

For different culture volumes the following parameters are used:

Surface Volume Optional area plating Dilution DNA Transfection Plus ™Culture per well medium Volume quantity composition Reagent Vessel (cm²)(μL) (μL) (ng) (μL) (μL) 96-well 0.3 100 20 100 0.25 0.1 48-well 1.0 20040 200 0.5 0.2 24-well 2 500 100 500 1.25 0.5 12-well 4 1000 200 10002.5 1.0  6-well 10 2000 500 2500 6.25 2.5

Reverse Transfection

Rapid 96-well transfections are obtained by plating cells directly intothe transfection mix. Complexes are prepared in the plate and cells aredirectly added at twice the cell density as described above, in 100 μLvolume. More lipid is used for optimal transfection.Note: The transfection composition is tested for absence of microbialcontamination using blood agar plates, Sabraud dextrose agar plates andfluid thioglycolate medium and functionally by transfection with areporter plasmid.

Example 5 High-Throughput Protocol

The following protocol is used to transfect DNA (β-galactosidasereporter plasmid pCMV•SPORT-β-gal) into mammalian cells for higherthroughput or for using smaller amounts of transfection composition. Inthis procedure, the reagents are pre-diluted first, and then a largervolume is added to the diluted DNA. The amounts used are on a per wellbasis for a 96-well format.

Adherent Cells

One day before transfection, plate cells in 100 μL of growth medium sothat the cells are 50-80% confluent at the time of transfection.

Suspension Cells

Just prior to preparation of the complexes, 40,000-100,000 cells areplated in 100 μL of growth medium.

Transfection

For each transfection sample the complexes are prepared as follows.

100 ng of plasmid DNA is diluted in 10 μL Opti-MEM® I Reduced SerumMedium without serum and is gently mixed. (For optional transfectionswith Plus™ Reagent (Invitrogen Inc., Carlsbad Calif.), the Plus™ Reagentis mixed gently, diluted 10 fold with Opti-MEM® I Reduced Serum Mediumwithout serum (0.1 μL per well), and 1 μL of diluted Plus™ Reagent isadded directly to the diluted DNA, is mixed gently and is incubated for5 minutes at room temperature.) Then a stock solution of transfectioncomposition is made by diluting 0.25 μL per well of a transfectioncomposition provided herein that includes a fusogenic peptide, acationic lipid, and a neutral lipid at a combined total concentration of1 mg/ml- to 2 mg/ml in Opti-MEM® I Reduced Serum Medium without serum togive 10 μL per well. Then 10 μL of a diluted a transfection compositionprovided herein that includes a fusogenic peptide, a neutral lipid, anda cationic lipid is added to the diluted DNA solution (with or withoutPlus™ Reagent) and the mixture is gently mixed and is incubated for 30minutes at room temperature to form the complexes. Then approximately 20μL of the complex is added directly to each well containing the cellsand the plate is gently rocked back and forth. Cells are incubated at37° C. in a CO₂ incubator for an additional 18-48 hours prior to a testfor transgene expression. Medium is optionally changed after 4-6 hours.

Medium is removed and the cells are lysed in 100-200 μl of lysis buffer.The lysates (20 μl) are assayed for β-gal activity using the enzymaticsubstrate ONPG. Total activity was determined by reading the OD at 405nm using a Bio-Rad Benchmark Microplate Spectrophotometer.

For Optimizing Transfections, Generating Stable Cells Lines and ScalingUp or DownTransfections see Example 4.

All compounds in the Examples below were characterized by massspectrometry and the mass spectra conformed to the expected formulae.

Example 6 Synthesis of1,4-Bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine

piperazine (2.0 g), N-(2,3-epoxypropyl)phthalmide (12.0 g) and lithiumperchlorate (6.4 g) were combined in 300 ml absolute ethanol and heatedunder reflux for 2 days. The resulting mix was cooled, and theprecipitate obtained by filtration was rinsed with ethanol to provide1,4,-bis(2-hydroxy-3-phthalimidopropyl)piperizine (Compound 1), as anoff-white solid (8.5 g, 75% yield).

Hydrazine hydrate (4 ml) was added to a solution of Compound 1 (8.5 g)in 200 ml of ethanol. The reaction mix was heated under reflux overnightand cooled to room temperature for about 1 hr and the precipitatefiltered. The precipitate was rinsed with ethanol and used furtherwithout purification. The compound that was obtained,1,4-bis(3-amino-2-hydroxypropyl)piperazine (Compound 2) was acylatedusing oleoyl chloride followed by reduction with lithium aluminumhydride. To an ice cooled solution of compound 2 in THF (400 ml) wasadded 15 g of oleoyl chloride and 15 ml of DIPEA. The reaction mix wasstirred overnight under reflux. The reaction mixture was diluted withchloroform (400 ml) and sequentially extracted with water (2×200 ml),10% HCl (300 ml) and 0.2% KOH (200 ml). The organic solvent was removedon a rotary evaporator and the resulting compound dried overnight invacuo.

The crude diamide compound was suspended in anhydrous THF (400 ml) and100 ml of a 1M lithium aluminium hydride solution in THF was addeddrop-wise. After the addition was completed, the reaction mix wasrefluxed overnight. More THF (150 ml) was added and the reaction mix wascooled to room temperature. A 15% sodium hydroxide solution (200 ml) wasadded drop-wise to the mixture and stirred for 2 hours. The THF layerwas decanted and the remaining suspension was exhaustively extractedwith chloroform, using TLC to monitor the presence of the desiredproduct in the chloroform layer. The THF and chloroform layers werecombined and evaporated to obtain the desired compound,octadec-9-enyl-{2-hydroxy-3-[4-(3-octadec-9-enylamino-2-hydroxypropyl)-piperazin-1-yl]-propyl}-amine(Compound 3) (3.4 g). The product was characterized by massspectrometry.

Compound 3 (2.5 g) was treated with N-(3-bromopropyl)phthalimide (2.19g) and diisopropylethylamine (2 ml) in DMF (10 ml). The reaction mixturewas heated at 100° C. for 3 hours and then diluted with chloroform (300ml) and extracted with water (4×300 ml). The chloroform was removed on arotary evaporator and the residue subjected to flash chromatographyusing chloroform and methanol/chloroform as eluants to obtain thedesired phthlamide adduct (Compound 4). Hydrazine hydrate (0.75 ml) wasadded to a solution of the phthalimide compound 4 (1.7 g) in 100%reagent alcohol (100 ml). The reaction mix was refluxed overnight andcooled to room temperature for about 1 hr. The reaction mix was thencooled at 4° C. overnight and the precipitate obtained by filtration.The solid was washed with ethanol chilled to −20° C. (2×20 ml) and driedin vacuo. The residue was dissolved in chloroform (300 ml), filtered andextracted twice with water (200 ml). The chloroform was removed on arotary evaporator to give1,4-Bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine(Compound 5) in a quantitative yield. This material was acidified withHCL in dioxane and purified on reverse phase (C-18) flash chromatographyusing aqueous methanol as eluant and characterized by TLC and massspectrometry.

In this manner compounds with alkyl groups varying in length from C₁₂ toC₁₈ were synthesized. Examples include compounds where R₁, R₂═H; X₁,X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; W₁, W₂═H; q, p, m=1;Y=piperazine; and Z₁ and Z₂ both are palmityl, myristyl; lauryl; orstearyl.

Example 7 Synthesis of1,4-Bis[(3-(3-amino-2-hydroxypropyl)-oleoylamino)-2-hydroxypropyl]piperazine

Compound 3,octadec-9-enyl-{2-hydroxy-3-[4-(3-octadec-9-enylamino-2-hydroxypropyl)-piperazin-1-yl]-propyl}-amine(3.2 g) was dissolved in ethanol (110 ml), andN-(2,3-epoxypropyl)phthalmide (2.3 g) and lithium perchlorate (1.1 g)were added to the reaction mix which was then heated under refluxovernight. The reaction mix was cooled and diluted with chloroform (300ml) and extracted with water (2×300 ml). The organic phase wasconcentrated and subjected to flash chromatography usingchloroform/methanol (1-3%) as eluant to provide the desired phthalamide(3.05 g)

Hydrazine hydrate (0.7 ml) was added to a solution of the phthalimide(3.0 g) in 100 ml of ethanol. The reaction mix was refluxed overnightand cooled to room temperature for about 1 hr and the precipitatefiltered. The filtrate was diluted with chloroform (300 ml) andextracted with water (2×300 ml). The organic layer was concentrated andthe material thus obtained with acidified using HCL in dioxane andsubjected to flash chromatography on a C₁₈ reversed-phase column. Thedesired compound,1,4-Bis[(3-(3-amino-2-hydroxypropyl)-oleoylamino)-2-hydroxypropyl]piperazine,was obtained as a solid (2.32 g) (R₁, R₂═H; X₁, X₂═CH₂; R₄,R₅═CH₂—CHOH—CH₂; R₃, R₆═N; Z₁, Z₂=oleoyl; W₁, W₂═OH; q, p, m=1; andY=piperazine). In this manner compounds with alkyl groups varying inlength from C₁₂ to C₁₈ were synthesized. Examples include compoundswhere:

R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CHOH—CH₂; R₃, R₆═N; W₁, W₂═OH; q, p,m=1; Y=piperazine; and Z₁ and Z₂ both are palmityl, myristyl; lauryl; orstearyl.

Example 8 Synthesis of1,4-Bis[(3-(3-amino-2-hydroxypropyl)-oleoylamino)-propyl]piperazine

To an ice cooled solution of 1,4-bis(3-aminopropyl)piperazine (10.0 g)in THF (200 ml) was added 38 g of technical oleoyl chloride (85%). Thereaction mix was stirred overnight at room temperature. The reactionmixture was diluted with chloroform (500 ml) and extracted withsaturated sodium bicarbonate solution (3×200 ml). The organic solventwas removed on a rotary evaporator and the residue was subjected toshort column chromatography on silica gel using chloroform and 5-20%methanol/chloroform as eluants. The fractions containing the desiredcompound, octadec-9-enoicacid{3-[4-(3-octadec-9-enoylamino-propyl)-piperizine-1-yl]-propyl}-amide,were combined and concentrated to obtain an off-white solid (18.28 g,50% yield). The product was characterized by mass spectrometry.

The diamide compound, octadec-9-enoicacid{3-[4-(3-octadec-9-enoylamino-propyl)-piperizine-1-yl]-propyl}-amide(6.2 g) was suspended in anhydrous THF (150 ml) and 100 ml of 1 Mlithium aluminium hydride solution in THF was added drop-wise. After theaddition was completed, the reaction mix was heated under refluxovernight. More THF (150 ml) was added and the reaction mix was cooledto room temperature. A 15% sodium hydroxide solution (100 ml) was addeddrop-wise to the mixture and stirred for 2 hours. A saturated sodiumbicarbonate solution (250 ml) was added and stirred for approximately 1hour. The THF layer was decanted and the remaining suspension wasexhaustively extracted with chloroform, using TLC to monitor thepresence of the desired product in the chloroform layer. The THF andchloroform layers were combined and evaporated to obtain the desiredcompound,octadec-9-enyl-{3-[4-(3-octadec-9-enylamino-propyl)-piperazin-1-yl]-propyl}-amine(5.11 g, 85% yield). The product was characterized by mass spectrometry.The amine,octadec-9-enyl-{3-[4-(3-octadec-9-enylamino-propyl)-piperazin-1-yl]-propyl}-amine(5.0 g, 7.1 mmol) was treated with N-(2,3-epoxypropyl)-phthalimide (3.6g, 17.7 mmol) and lithium perchlorate (1.8 g, 17.0 mmol) in 150 mlreagent alcohol. The mixture was refluxed overnight, cooled and dilutedwith 400 ml chloroform. The chloroform solution was extracted twice withwater (300 ml). The chloroform was removed on a rotary evaporator toobtain the bis-phthalimide adduct as a gum, which was purified by flashchromatography on silica using 1% ethanol in chloroform as eluant, toprovide 2.3 g (30% yield) of pure material. The compound wascharacterized by mass spectrometry.

Hydrazine hydrate (0.5 ml) was added to a solution of the phthalimide(2.25 g) in 100% reagent alcohol (100 ml). The reaction mix was refluxedovernight and cooled to room temperature for about 1 hr. The reactionmix was then cooled at 4° C. overnight and the precipitate obtained byfiltration. The solid was washed twice with ethanol chilled to −20° C.(20 ml). The ethanol was removed on a rotary evaporator and theresulting solid dissolved in 300 ml chloroform, filtered and extractedtwice with 200 ml water. The chloroform was removed on a rotaryevaporator to give1,4-Bis[(3-(3-amino-2-hydroxypropyl)-oleoylamino)-propyl]piperazine in aquantitative yield (R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N;Z₁, Z₂=oleoyl; W₁, W₂═OH; q, p, m=1; and Y=piperazine). This materialwas acidified with HCL in dioxane and purified on reverse phase (C-18)flash chromatography using aqueous methanol as eluant and characterizedby TLC and mass spectrometry. In this manner compounds with alkyl groupsvarying in length from C₁₂ to C₁₈ were synthesized. Specifically thefollowing additional compounds were synthesized. Examples includecompounds where: R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; W₁,W₂═OH; q, p, m=1; Y=piperazine; and Z₁ and Z₂ both are palmityl,myristyl, lauryl or stearyl.

Example 9 Synthesis of1,4-Bis[(3-(3-aminopropyl)-oleoylamino)propyl]piperazine

The amine,octadec-9-enyl-{3-[4-(3-octadec-9-enylamino-propyl)-piperazin-1-yl]-propyl}-amine(5 g, 7.1 mmol) above was treated with N-(3-bromopropyl)phthalimide(6.78 g, 25 mmol) and diisopropylethylamine (3.7 ml, 21 mmol) in DMF (50ml). The reaction mixture was heated to 120° C. for 1 hour and then at95° C. overnight. The solvent was removed by rotary evaporation and thedesired phthalamide was isolated by silica flash chromatography usingchloroform and methanol/chloroform as eluants. The phthalimide wastreated with hydrazine hydrate as described above to obtain1,4-Bis[(3-(3-aminopropyl)-oleoylamino)propyl]piperazine (R₁, R₂═H; X₁,X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; Z₁, Z₂=oleoyl; W₁, W₂═H; q, p,m=1; and Y=piperazine). The compound was characterized by TLC and massspectrometry. In this manner compounds with alky groups varying inlength from C₁₂ to C₁₈ were synthesized. Examples include compoundswhere: R₁, R₂═H; X₁, X₂═CH₂; R₄, R₅═CH₂—CH₂—CH₂; R₃, R₆═N; W₁, W₂═H; q,p, m=1; Y=piperazine; and Z₁ and Z₂ both are palmityl, myristyl, laurylor stearyl.

Example 10 Synthesis of1-amino-3-[(3-amino-2-hydroxy-propyl)-octadec-9-enyl-amino]-propan-2-ol

Oleylamine (2.67 g, 10 mmol) was treated with excessN-(2,3-epoxy-propyl)phthalimide (10.72, 40 mmol) anddiisopropylethylamine (6.7 ml) in DMF (50 ml). The reaction mix washeated at 95° C. overnight. The solvent was removed by rotaryevaporation and the resulting gum was taken up in chloroform (200 ml)and extracted twice with water (200 ml). The chloroform was removed byevaporation and the resulting material subjected to short columnchromatography over silica using chloroform as eluant. The desiredmaterial thus obtained was treated with hydrazine hydrate as above toobtain1-amino-3-[(3-amino-2-hydroxy-propyl)-octadec-9-enyl-amino]-propan-2-ol(Compound 6).

Example 11 Formulation of Cationic Lipids into Liposomes

In general, the required amount of the cationic lipid and the co-lipidare weighed and transferred into a round bottom flask. An amount ofchloroform that is sufficient to dissolve the lipids is added, followedby sufficient molecular biology-grade water to produce the desiredconcentration of total lipids/volume (e.g. 2 mg/ml). The chloroform isremoved under vacuum in a rotary evaporator. As the chloroform isremoved, liposomes are formed in the aqueous medium. The solutionbecomes opalescent and varies in its turbidity depending on the cationiclipid and co-lipid being formulated

More specifically, the HCl salt of1,4-Bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine wasformulated without a co-lipid and using DOPE or cholesterol asco-lipids. Thus, 14.9 mg of the cationic lipid and 5.1 mg of DOPE (M/M,2:1) were dissolved in 1 ml chloroform. To the chloroform solution 10 mlof water was added and the heterogeneous solution was evaporated undervacuum on the rotary evaporator. The chloroform was removed leaving aclear opalescent homogenous aqueous solution. The volume was readjustedto 10 ml to obtain a 2 mg/ml solution (129-A). In this manner, the HClsalt of1,4-Bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine wasformulated with DOPE in a M/M ratio of 4:1 (129-B), with cholesterol inM/M ratio of 2:1 (129-C), 4:1 (129-D) and 1:1 (130-H), and without aco-lipid (129-E).

Example 12 Transfection Protocol

Transfection of CHO-K1 and HEK293 with β-galactosidase reporter plasmidpCMV•SPORT-β-gal was carried out as follows:

Cells were plated in 96-well plates with 100 μl of media containing 10%fetal calf serum the day before transfection such that a desiredconfluency (70%-95%) was achieved.

The following day, lipid and DNA were mixed in Opti-MEM medium to formDNA/lipid complexes. Complexes were formed by adding various amounts oflipids (0.1 to 0.35 μl) to 100 μl of Opti-MEM. DNA (100 ng) was added to100 μl Opti-MEM. The DNA and lipids solutions were then mixed to formDNA-lipid complexes. The complexes were incubated for at least 20-30minutes and 20 μl of the complexes was added directly to the cells in10% serum. Cells were incubated for an additional 24 hours to allowexpression of the plasmid. Medium was removed and the cells were lysedin 100-200 μl of lysis buffer. The lysates (20 μl) were assayed forβ-gal activity using the enzymatic substrate ONPG. Total activity wasdetermined by reading the OD at 405 nm using a Bio-Rad BenchmarkMicroplate Spectrophotometer.

Example 13 siRNA Transfection

For siRNA transfection, a 24 well plate is seeded with the appropriatenumber of cells in serum containing medium a day before transfectionsuch that they are 50 to 60% confluent, and the cells are incubated at37° C. in a 3-5% CO₂ incubator overnight. For each well to betransfected, 25 μl of serum free medium containing 0.1 to 0.4 μl oflipid and 25 μl of serum-free medium containing siRNA is prepared. Finalconcentration of siRNA is 10 nM. The lipid and siRNA solutions are mixedand incubated at room temperature for 20 minutes. The lipid/siRNAcomplex (50 μl) is added to the cells in serum-containing medium and thecells are incubated at 37° C. in a CO₂ incubator. Gene silencing can bemonitored at 24 to 72 hours after transfection.

1. A complex comprising a transfection agent and a fusion agent, whereinsaid fusion agent comprises a fusion promoting amino acid sequencederived from a fusion protein of a non-enveloped virus.
 2. The complexaccording to claim 1, wherein said non-enveloped virus is a Reovirus. 3.The complex according to claim 1, wherein said Reovirus is selected fromthe group consisting of Avian Reovirus, Nelson Bay Reovirus, PulauReovirus, and Baboon reovirus.
 4. The complex according to claim 1,wherein said fusion agent further comprises a nucleic acid bindingmoiety functionally linked to said fusion promoting amino acid sequence.5-32. (canceled)
 33. A method for introducing a nucleic acid, protein,or peptide into a cell, comprising contacting the cell with a complexcomprising the nucleic acid, protein, or peptide a transfection agent,and a fusion agent, wherein said fusion agent comprises a fusionpromoting amino acid sequence derived from a fusion protein of anon-enveloped virus, thereby introducing the nucleic acid, protein, orpeptide into the cell. 34-45. (canceled)
 46. A kit comprising a nucleicacid binding moiety and a peptide or protein or a modified peptide ormodified protein derived from a fusion promoting amino acid sequence ofa Reovirus.
 47. The kit of claim 46 further comprising a cationic lipidtransfection agent. 48-52. (canceled)
 53. A peptide of between 20 and 50amino acids in length, comprising a fusogenic peptide derived from anon-enveloped virus.
 54. The peptide according to claim 53 wherein saidnucleic acid binding moiety is a polycationic peptide. 55-58. (canceled)59. A fusogenic peptide according to claim 53, wherein said peptidecomprises 5-30 contiguous amino acids of a sequence selected from thegroup consisting of: (SEQ ID NO: 1)MLRMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA, (SEQ ID NO: 2)MPRMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIA, (SEQ ID NO: 3)MSGDCAGLVSVFGSVHCQSSKNKAGGDLQATSILTTYWPH, (SEQ ID NO: 4)MSSDCAKIVSVFGSVHCQSSKNSAGGDLQATSVFTTYWPH, (SEQ ID NO: 6)MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVI, and (SEQ ID NO: 5)MGQRHSIVQPPAPPPNAFVEIVSSSTGIIIAVGIFAFIFS,


60. A peptide according to claim 59, wherein said peptide comprises atleast 10 contiguous amino acids of an amino acid sequence selected fromthe group consisting of: RMPPGSCNGATAVFGNVH (SEQ ID NO: 7)

RMPPGSCNGATAIFGNVH (SEQ ID NO: 23) GDCAGLVSVFGSVH (SEQ ID NO: 8)SDCAKIVSVFGSVH (SEQ ID NO: 9) SGPSNFVNHAPGEAIVT (SEQ ID NO: 11)QRHSIVQPPAPPPNAFVEIVS (SEQ ID NO: 10) and SGPSNFVNHAPGEAIVT,(SEQ ID NO: 11)

covalently linked to between 8 and 30 lysine residues. 61-62. (canceled)63. A kit according to claim 46, wherein the peptide or protein or themodified peptide or modified protein, is a fusogenic peptide from AvianReovirus, Nelson Bay Reovirus, or Pulau Reovirus.
 64. A kit according toclaim 46, wherein the kit further comprises a transfection enhancingagent. 65-83. (canceled)
 84. The method of claim 33, wherein thetransfection agent comprises a cationic lipid.
 85. The method of claim33, wherein the transfection agent further comprises a neutral lipid.86. (canceled)
 87. The method of claim 33, wherein the fusion promotingamino acid sequence is a fusogenic peptide that is between 20 and 50amino acids in length. 88-114. (canceled)
 115. A lipid having theformula:

wherein X₁ and X₂ independently are selected from the group consistingof (CH₂)_(n), (CHOH)_(n), and CONH; X₅ and X₆ independently are(CH₂)₁₋₆; W₁ and W₂ independently are selected from the group consistingof, hydrogen, —OH, —O—(C₁-C₁₈)alkyl, —O—(C₁-C₁₈)alkenyl,—O—(C₁-C₁₈)alkynyl, —NH₂, —NH(CH₂)_(s)CH₃, —N((CH₂)_(s)CH₃), —SH, and—NH—NH₂; R₃ and (R₆)_(q) independently are selected from the groupconsisting of N, NH, CH, N(CH₂)_(s)CH₃, (CH)_(n), (COH)_(n), CON— andq=0-1; R₄ and R₅ independently are selected from the group consisting of(CH₂)_(n), (CH₂—CHOH—CH₂)_(n), (CHOH)_(n), HNCO, CONH, CO, —O—, —S—,—S—S—, polyamide and an ester linkage; L₁ and L₂ independently areselected from the group consisting of —NH—, —O—, —NHCO—, —CONH—, —OCO—,—COO—, —CO—, —S—, —S—S—, —NHC(O)O—, —OC(O)NH—, —NHCONH—, —NHC(═NH)NH—,—NH—NH—, —S(O)— and —SO₂—; Y is a heterocyclic moiety containing atleast one amine or amide moiety, wherein the points of attachment of Yare carbon and/or heteroatoms; R₁ and R₂ independently are selected fromthe group consisting of hydrogen, primary alkylamine, secondaryalkylamine, tertiary alkyl amine, quaternary alkylamine, alkenylamine,secondary alkenylamine, tertiary alkenyl amine, quaternary alkenylamine,alkynylamine, secondary alkynylamine, tertiary alkynylamine, quaternaryalkynylamine amino alcohol, alkyl polyamine, alkenyl polyamine, alkynylpolyamine, spermidine, spermine, carboxy spermine, guanidinium,pyridinium, pyrollidinium, piperidinium, piperazinium, amino acyl,peptidyl, and protein; Z₁ and Z₂ independently are selected from thegroup consisting of straight chain alkyl, branched alkyl, cycloalkyl,straight chain alkenyl, branched alkenyl, cycloalkenyl, straight chainalkynyl, and branched alkynyl, m, n, p, and s independently are 0-6,with the proviso that when m, n, and p all are 0 then Y is eliminatedand R₃ is bonded directly to X₂. 116-117. (canceled)
 118. A lipidaccording to claim 115 having the formula:

wherein X₁, X₂, W₁, W₂, R₃, (R₆), R₄, R₅, Y, R₁, R₂, Z₁, Z₂ m, n, p, ands are as defined above.
 119. A lipid according to claim 115 wherein Y is

wherein X₃ and X₄ are independently selected from N and CH and whereinn₁ and n₂ independently are 1-10
 120. A lipid according to claim 115,wherein X₃ and X₄ are N and n₁ and n₂ independently are 1-10 121-162.(canceled)